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SPIM - Light Sheet Microscopy Literature Database

Below you find a list of publications on the theory, design and applications of light sheet microscopy and similar techniques. I try to keep this list up-to-date, but please email me if your paper is missing! Latest update: September 24, 2015

PDFs are available on request.

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Abbott, A. Microscopic marvels: Seeing the system 2009 Nature
Vol. 459(7247), pp. 630-631 
article DOI URL 
BibTeX:
@article{Abbott2009-ds,
  author = {Abbott, Alison},
  title = {Microscopic marvels: Seeing the system},
  journal = {Nature},
  year = {2009},
  volume = {459},
  number = {7247},
  pages = {630--631},
  url = {http://dx.doi.org/10.1038/459630a},
  doi = {http://dx.doi.org/10.1038/459630a}
}
Adams, M.W., Loftus, A.F., Dunn, S.E., Joens, M.S. and Fitzpatrick, J.A.J. Light Sheet Fluorescence Microscopy (LSFM): Light Sheet
Fluorescence Microscopy (LSFM)
2001 Current Protocols in Cytometry, pp. 12.37.1-12.37.15  incollection DOI URL 
Abstract: The development of confocal microscopy techniques introduced the
ability to optically section fluorescent samples in the axial
dimension, perpendicular to the image plane. These approaches,
via the placement of a pinhole in the conjugate image plane,
provided superior resolution in the axial (z) dimension
resulting in nearly isotropic optical sections. However,
increased axial resolution, via pinhole optics, comes at the
cost of both speed and excitation efficiency. Light sheet
fluorescent microscopy (LSFM), a century-old idea made possible
with modern developments in both excitation and detection
optics, provides sub-cellular resolution and optical sectioning
capabilities without compromising speed or excitation
efficiency. Over the past decade, several variations of LSFM
have been implemented each with its own benefits and
deficiencies. Here we discuss LSFM fundamentals and outline the
basic principles of several major light-sheet-based imaging
modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam
SPIM, and stimulated emission depletion SPIM) while considering
their biological relevance in terms of intrusiveness, temporal
resolution, and sample requirements. pyright 2015 by John
Wiley & Sons, Inc.
BibTeX:
@incollection{Adams2001-vk,
  author = {Adams, Michael W and Loftus, Andrew F and Dunn, Sarah E and
Joens, Matthew S and Fitzpatrick, James A J}, title = {Light Sheet Fluorescence Microscopy (LSFM): Light Sheet
Fluorescence Microscopy (LSFM)}, booktitle = {Current Protocols in Cytometry}, publisher = {John Wiley & Sons, Inc.}, year = {2001}, pages = {12.37.1--12.37.15}, url = {http://doi.wiley.com/10.1002/0471142956.cy1237s71}, doi = {http://dx.doi.org/10.1002/0471142956.cy1237s71} }
Ahrens, M.B., Orger, M.B., Robson, D.N., Li, J.M. and Keller, P.J. Whole-brain functional imaging at cellular resolution using
light-sheet microscopy
2013 Nat. Methods
Vol. 10(5), pp. 413-420 
article DOI URL 
Abstract: Brain function relies on communication between large
populations of neurons across multiple brain areas, a full
understanding of which would require knowledge of the
time-varying activity of all neurons in the central nervous
system. Here we use light-sheet microscopy to record activity,
reported through the genetically encoded calcium indicator
GCaMP5G, from the entire volume of the brain of the larval
zebrafish in vivo at 0.8 Hz, capturing more than 80% of all
neurons at single-cell resolution. Demonstrating how this
technique can be used to reveal functionally defined circuits
across the brain, we identify two populations of neurons with
correlated activity patterns. One circuit consists of
hindbrain neurons functionally coupled to spinal cord
neuropil. The other consists of an anatomically symmetric
population in the anterior hindbrain, with activity in the
left and right halves oscillating in antiphase, on a timescale
of 20 s, and coupled to equally slow oscillations in the
inferior olive.
BibTeX:
@article{Ahrens2013-rs,
  author = {Ahrens, Misha B and Orger, Michael B and Robson, Drew N and
Li, Jennifer M and Keller, Philipp J}, title = {Whole-brain functional imaging at cellular resolution using
light-sheet microscopy}, journal = {Nat. Methods}, year = {2013}, volume = {10}, number = {5}, pages = {413--420}, url = {http://dx.doi.org/10.1038/nmeth.2434}, doi = {http://dx.doi.org/10.1038/nmeth.2434} }
Alvers, A.L., Ryan, S., Scherz, P.J., Huisken, J. and Bagnat, M. Single continuous lumen formation in the zebrafish gut is
mediated by smoothened-dependent tissue remodeling
2014 Development
Vol. 141(5), pp. 1110-1119 
article DOI URL 
Abstract: The formation of a single lumen during tubulogenesis is
crucial for the development and function of many organs.
Although 3D cell culture models have identified molecular
mechanisms controlling lumen formation in vitro, their
function during vertebrate organogenesis is poorly understood.
Using light sheet microscopy and genetic approaches we have
investigated single lumen formation in the zebrafish gut. Here
we show that during gut development multiple lumens open and
enlarge to generate a distinct intermediate, which consists of
two adjacent unfused lumens separated by basolateral contacts.
We observed that these lumens arise independently from each
other along the length of the gut and do not share a
continuous apical surface. Resolution of this intermediate
into a single, continuous lumen requires the remodeling of
contacts between adjacent lumens and subsequent lumen fusion.
We show that lumen resolution, but not lumen opening, is
impaired in smoothened (smo) mutants, indicating that
fluid-driven lumen enlargement and resolution are two distinct
processes. Furthermore, we show that smo mutants exhibit
perturbations in the Rab11 trafficking pathway and demonstrate
that Rab11-mediated trafficking is necessary for single lumen
formation. Thus, lumen resolution is a distinct genetically
controlled process crucial for single, continuous lumen
formation in the zebrafish gut.
BibTeX:
@article{Alvers2014-un,
  author = {Alvers, Ashley L and Ryan, Sean and Scherz, Paul J and
Huisken, Jan and Bagnat, Michel}, title = {Single continuous lumen formation in the zebrafish gut is
mediated by smoothened-dependent tissue remodeling}, journal = {Development}, year = {2014}, volume = {141}, number = {5}, pages = {1110--1119}, url = {http://dx.doi.org/10.1242/dev.100313}, doi = {http://dx.doi.org/10.1242/dev.100313} }
Amat, F. and Keller, P.J. Towards comprehensive cell lineage reconstructions in complex
organisms using light-sheet microscopy
2013 Dev. Growth Differ.
Vol. 55(4), pp. 563-578 
article DOI URL 
Abstract: Understanding the development of complex multicellular
organisms as a function of the underlying cell behavior is one
of the most fundamental goals of developmental biology. The
ability to quantitatively follow cell dynamics in entire
developing embryos is an indispensable step towards such a
system-level understanding. In recent years, light-sheet
fluorescence microscopy has emerged as a particularly
promising strategy for recording the in vivo data required to
realize this goal. Using light-sheet fluorescence microscopy,
entire complex organisms can be rapidly imaged in three
dimensions at sub-cellular resolution, achieving high temporal
sampling and excellent signal-to-noise ratio without damaging
the living specimen or bleaching fluorescent markers. The
resulting datasets allow following individual cells in
vertebrate and higher invertebrate embryos over up to several
days of development. However, the complexity and size of these
multi-terabyte recordings typically preclude comprehensive
manual analyses. Thus, new computational approaches are
required to automatically segment cell morphologies,
accurately track cell identities and systematically analyze
cell behavior throughout embryonic development. We review
current efforts in light-sheet microscopy and bioimage
informatics towards this goal, and argue that comprehensive
cell lineage reconstructions are finally within reach for many
key model organisms, including fruit fly, zebrafish and mouse.
BibTeX:
@article{Amat2013-mr,
  author = {Amat, Fernando and Keller, Philipp J},
  title = {Towards comprehensive cell lineage reconstructions in complex
organisms using light-sheet microscopy}, journal = {Dev. Growth Differ.}, year = {2013}, volume = {55}, number = {4}, pages = {563--578}, url = {http://dx.doi.org/10.1111/dgd.12063}, doi = {http://dx.doi.org/10.1111/dgd.12063} }
Amat, F., Lemon, W., Mossing, D.P., McDole, K., Wan, Y., Branson, K., Myers, E.W. and Keller, P.J. Fast, accurate reconstruction of cell lineages from
large-scale fluorescence microscopy data
2014 Nat. Methods
Vol. 11(9), pp. 951-958 
article DOI URL 
Abstract: The comprehensive reconstruction of cell lineages in complex
multicellular organisms is a central goal of developmental
biology. We present an open-source computational framework for
the segmentation and tracking of cell nuclei with high
accuracy and speed. We demonstrate its (i) generality by
reconstructing cell lineages in four-dimensional,
terabyte-sized image data sets of fruit fly, zebrafish and
mouse embryos acquired with three types of fluorescence
microscopes, (ii) scalability by analyzing advanced stages of
development with up to 20,000 cells per time point at 26,000
cells min(-1) on a single computer workstation and (iii) ease
of use by adjusting only two parameters across all data sets
and providing visualization and editing tools for efficient
data curation. Our approach achieves on average 97.0% linkage
accuracy across all species and imaging modalities. Using our
system, we performed the first cell lineage reconstruction of
early Drosophila melanogaster nervous system development,
revealing neuroblast dynamics throughout an entire embryo.
BibTeX:
@article{Amat2014-qb,
  author = {Amat, Fernando and Lemon, William and Mossing, Daniel P and
McDole, Katie and Wan, Yinan and Branson, Kristin and Myers,
Eugene W and Keller, Philipp J}, title = {Fast, accurate reconstruction of cell lineages from
large-scale fluorescence microscopy data}, journal = {Nat. Methods}, publisher = {Nature Publishing Group}, year = {2014}, volume = {11}, number = {9}, pages = {951--958}, url = {http://dx.doi.org/10.1038/nmeth.3036}, doi = {http://dx.doi.org/10.1038/nmeth.3036} }
Amat, F., Myers, E.W. and Keller, P.J. Fast and robust optical flow for time-lapse microscopy using
super-voxels
2013 Bioinformatics
Vol. 29(3), pp. 373-380 
article DOI URL 
Abstract: MOTIVATION: Optical flow is a key method used for quantitative
motion estimation of biological structures in light
microscopy. It has also been used as a key module in
segmentation and tracking systems and is considered a mature
technology in the field of computer vision. However, most of
the research focused on 2D natural images, which are small in
size and rich in edges and texture information. In contrast,
3D time-lapse recordings of biological specimens comprise up
to several terabytes of image data and often exhibit complex
object dynamics as well as blurring due to the
point-spread-function of the microscope. Thus, new approaches
to optical flow are required to improve performance for such
data. RESULTS: We solve optical flow in large 3D time-lapse
microscopy datasets by defining a Markov random field (MRF)
over super-voxels in the foreground and applying motion
smoothness constraints between super-voxels instead of
voxel-wise. This model is tailored to the specific
characteristics of light microscopy datasets: super-voxels
help registration in textureless areas, the MRF over
super-voxels efficiently propagates motion information between
neighboring cells and the background subtraction and
super-voxels reduce the dimensionality of the problem by an
order of magnitude. We validate our approach on large 3D
time-lapse datasets of Drosophila and zebrafish development by
analyzing cell motion patterns. We show that our approach is,
on average, 10 faster than commonly used optical
flow implementations in the Insight Tool-Kit (ITK) and reduces
the average flow end point error by 50% in regions with
complex dynamic processes, such as cell divisions.
AVAILABILITY: Source code freely available in the Software
section at http://janelia.org/lab/keller-lab.
BibTeX:
@article{Amat2013-be,
  author = {Amat, Fernando and Myers, Eugene W and Keller, Philipp J},
  title = {Fast and robust optical flow for time-lapse microscopy using
super-voxels}, journal = {Bioinformatics}, year = {2013}, volume = {29}, number = {3}, pages = {373--380}, url = {http://dx.doi.org/10.1093/bioinformatics/bts706}, doi = {http://dx.doi.org/10.1093/bioinformatics/bts706} }
Andilla, J., Olarte, O.E., Aviles-Espinosa, R. and Loza-Alvarez, P. Imaging deep and clear in thick inhomogeneous samples 2014 SPIE MOEMS-MEMS, pp. 89780B-89780B-7  inproceedings DOI URL 
Abstract: abstract Acquisition of images deep inside large samples is one
of the most demanded improvements that current biology
applications ask for. Absorption, scattering and optical
aberrations are the main difficulties encountered in these types
of samples. ...
BibTeX:
@inproceedings{Andilla2014-lc,
  author = {Andilla, Jordi and Olarte, Omar E and Aviles-Espinosa, Rodrigo
and Loza-Alvarez, Pablo}, title = {Imaging deep and clear in thick inhomogeneous samples}, booktitle = {SPIE MOEMS-MEMS}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {89780B--89780B--7}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1842172}, doi = {http://dx.doi.org/10.1117/12.2041448} }
Annibale, P., Dvornikov, A. and Gratton, E. Electrically tunable lens speeds up 3D orbital tracking 2015 Biomed. Opt. Express
Vol. 6(6), pp. 2181-2190 
article DOI URL 
Abstract: 3D orbital particle tracking is a versatile and effective
microscopy technique that allows following fast moving
fluorescent objects within living cells and reconstructing
complex 3D shapes using laser scanning microscopes. We
demonstrated notable improvements in the range, speed and
accuracy of 3D orbital particle tracking by replacing commonly
used piezoelectric stages with Electrically Tunable Lens (ETL)
that eliminates mechanical movement of objective lenses. This
allowed tracking and reconstructing shape of structures
extending 500 microns in the axial direction. Using the ETL,
we tracked at high speed fluorescently labeled genomic loci
within the nucleus of living cells with unprecedented temporal
resolution of 8ms using a 1.42NA oil-immersion objective. The
presented technology is cost effective and allows easy upgrade
of scanning microscopes for fast 3D orbital tracking.
BibTeX:
@article{Annibale2015-bx,
  author = {Annibale, Paolo and Dvornikov, Alexander and Gratton, Enrico},
  title = {Electrically tunable lens speeds up 3D orbital tracking},
  journal = {Biomed. Opt. Express},
  year = {2015},
  volume = {6},
  number = {6},
  pages = {2181--2190},
  url = {http://dx.doi.org/10.1364/BOE.6.002181},
  doi = {http://dx.doi.org/10.1364/BOE.6.002181}
}
Ansari, N., Müller, S., Stelzer, E.H.K. and Pampaloni, F. Quantitative 3D cell-based assay performed with cellular
spheroids and fluorescence microscopy
2013 Methods Cell Biol.
Vol. 113, pp. 295-309 
article DOI URL 
Abstract: Cell-based assays are essential in both basic research and
drug discovery. Three-dimensional cellular spheroids are more
realistic models of tumors and healthy tissues compared to
standard two-dimensional cultures. Employing spheroids
improves the reliability and the physiological significance of
cell-based assays. We present a detailed drug assay protocol
performed with live cellular spheroids. We employ automated
epifluorescence live microscopy to investigate the effects of
drugs on the spheroids over several days. We describe the
spheroid preparation, manipulation, live fluorescence imaging,
and data processing. We quantify the autophagy-triggering
effects of the drugs (-)-Gossypol and Rapamycin in glioma cell
spheroids. The formation of the autophagosomes and the fusion
of the autophagosomes with lysosomes in the treated spheroids
are monitored over time and space with a mRFP:GFP:LC3 fusion
protein.
BibTeX:
@article{Ansari2013-ft,
  author = {Ansari, Nariman and Müller, Stefanie and Stelzer, Ernst
H K and Pampaloni, Francesco}, title = {Quantitative 3D cell-based assay performed with cellular
spheroids and fluorescence microscopy}, journal = {Methods Cell Biol.}, year = {2013}, volume = {113}, pages = {295--309}, url = {http://dx.doi.org/10.1016/B978-0-12-407239-8.00013-6}, doi = {http://dx.doi.org/10.1016/B978-0-12-407239-8.00013-6} }
Arnaout, R., Ferrer, T., Huisken, J., Spitzer, K., Stainier, D.Y.R., Tristani-Firouzi, M. and Chi, N.C. Zebrafish model for human long QT syndrome 2007 Proc. Natl. Acad. Sci. U. S. A.
Vol. 104(27), pp. 11316-11321 
article DOI URL 
Abstract: Long QT syndrome (LQTS) is a disorder of ventricular
repolarization that predisposes affected individuals to lethal
cardiac arrhythmias. To date, an appropriate animal model of
inherited LQTS does not exist. The zebrafish is a powerful
vertebrate model used to dissect molecular pathways of
cardiovascular development and disease. Because fundamental
electrical properties of the zebrafish heart are remarkably
similar to those of the human heart, the zebrafish may be an
appropriate model for studying human inherited arrhythmias.
Here we describe the molecular, cellular, and
electrophysiological basis of a zebrafish mutant characterized
by ventricular asystole. Genetic mapping and direct sequencing
identify the affected gene as kcnh2, which encodes the channel
responsible for the rapidly activating delayed rectifier K(+)
current (I(Kr)). We show that complete loss of functional
I(Kr) in embryonic hearts leads to ventricular cell membrane
depolarization, inability to generate action potentials (APs),
and disrupted calcium release. A small hyperpolarizing current
restores spontaneous APs, implying wild-type function of other
ionic currents critical for AP generation. Heterozygous fish
manifest overt cellular and electrocardiographic evidence for
delayed ventricular repolarization. Our findings provide
insight into the pathogenesis of homozygous kcnh2 mutations
and expand the use of zebrafish mutants as a model system to
study human arrhythmias.
BibTeX:
@article{Arnaout2007-qa,
  author = {Arnaout, Rima and Ferrer, Tania and Huisken, Jan and Spitzer,
Kenneth and Stainier, Didier Y R and Tristani-Firouzi, Martin
and Chi, Neil C}, title = {Zebrafish model for human long QT syndrome}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, year = {2007}, volume = {104}, number = {27}, pages = {11316--11321}, url = {http://dx.doi.org/10.1073/pnas.0702724104}, doi = {http://dx.doi.org/10.1073/pnas.0702724104} }
Arranz, A., Dong, D., Zhu, S., Rudin, M., Tsatsanis, C., Tian, J. and Ripoll, J. Helical optical projection tomography 2013 Opt. Express
Vol. 21(22), pp. 25912-25925 
article DOI URL 
Abstract: A new technique termed Helical Optical Projection Tomography
(hOPT) has been developed with the aim to overcome some of the
limitations of current 3D optical imaging techniques. hOPT is
based on Optical Projection Tomography (OPT) with the major
difference that there is a translation of the sample in the
vertical direction during the image acquisition process,
requiring a new approach to image reconstruction. Contrary to
OPT, hOPT makes possible to obtain 3D-optical images of intact
long samples without imposing limits on the sample length. This
has been tested using hOPT to image long murine tissue samples
such as spinal cords and large intestines. Moreover,
3D-reconstructed images of the colon of DSS-treated mice, a model
for Inflammatory Bowel Disease, allowed the identification of the
structural alterations. Finally, the geometry of the hOPT device
facilitates the addition of a Selective Plane Illumination
Microscopy (SPIM) arm, providing the possibility of delivering
high resolution images of selected areas together with complete
volumetric information.
BibTeX:
@article{Arranz2013-dy,
  author = {Arranz, Alicia and Dong, Di and Zhu, Shouping and Rudin, Markus
and Tsatsanis, Christos and Tian, Jie and Ripoll, Jorge}, title = {Helical optical projection tomography}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {22}, pages = {25912--25925}, url = {http://dx.doi.org/10.1364/OE.21.025912}, doi = {http://dx.doi.org/10.1364/OE.21.025912} }
Arrenberg, A.B., Stainier, D.Y.R., Baier, H. and Huisken, J. Optogenetic control of cardiac function 2010 Science
Vol. 330(6006), pp. 971-974 
article DOI URL 
Abstract: The cardiac pacemaker controls the rhythmicity of heart
contractions and can be substituted by a battery-operated
device as a last resort. We created a genetically encoded,
optically controlled pacemaker by expressing halorhodopsin and
channelrhodopsin in zebrafish cardiomyocytes. Using patterned
illumination in a selective plane illumination microscope, we
located the pacemaker and simulated tachycardia, bradycardia,
atrioventricular blocks, and cardiac arrest. The pacemaker
converges to the sinoatrial region during development and
comprises fewer than a dozen cells by the time the heart
loops. Perturbation of the activity of these cells was
entirely reversible, demonstrating the resilience of the
endogenous pacemaker. Our studies combine optogenetics and
light-sheet microscopy to reveal the emergence of organ
function during development.
BibTeX:
@article{Arrenberg2010-cp,
  author = {Arrenberg, Aristides B and Stainier, Didier Y R and Baier,
Herwig and Huisken, Jan}, title = {Optogenetic control of cardiac function}, journal = {Science}, year = {2010}, volume = {330}, number = {6006}, pages = {971--974}, url = {http://dx.doi.org/10.1126/science.1195929}, doi = {http://dx.doi.org/10.1126/science.1195929} }
Bagnat, M., Navis, A., Herbstreith, S., Brand-Arzamendi, K., Curado, S., Gabriel, S., Mostov, K., Huisken, J. and Stainier, D.Y.R. Cse1l is a negative regulator of CFTR-dependent fluid
secretion
2010 Curr. Biol.
Vol. 20(20), pp. 1840-1845 
article DOI URL 
Abstract: Transport of chloride through the cystic fibrosis
transmembrane conductance regulator (CFTR) channel is a key
step in regulating fluid secretion in vertebrates [1, 2]. Loss
of CFTR function leads to cystic fibrosis [1, 3, 4], a disease
that affects the lungs, pancreas, liver, intestine, and vas
deferens. Conversely, uncontrolled activation of the channel
leads to increased fluid secretion and plays a major role in
several diseases and conditions including cholera [5, 6] and
other secretory diarrheas [7] as well as polycystic kidney
disease [8-10]. Understanding how CFTR activity is regulated
in vivo has been limited by the lack of a genetic model. Here,
we used a forward genetic approach in zebrafish to uncover
CFTR regulators. We report the identification, isolation, and
characterization of a mutation in the zebrafish cse1l gene
that leads to the sudden and dramatic expansion of the gut
tube. We show that this phenotype results from a rapid
accumulation of fluid due to the uncontrolled activation of
the CFTR channel. Analyses in zebrafish larvae and mammalian
cells indicate that Cse1l is a negative regulator of
CFTR-dependent fluid secretion. This work demonstrates the
importance of fluid homeostasis in development and establishes
the zebrafish as a much-needed model system to study CFTR
regulation in vivo.
BibTeX:
@article{Bagnat2010-zv,
  author = {Bagnat, Michel and Navis, Adam and Herbstreith, Sara and
Brand-Arzamendi, Koroboshka and Curado, Silvia and Gabriel,
Sherif and Mostov, Keith and Huisken, Jan and Stainier, Didier
Y R}, title = {Cse1l is a negative regulator of CFTR-dependent fluid
secretion}, journal = {Curr. Biol.}, year = {2010}, volume = {20}, number = {20}, pages = {1840--1845}, url = {http://dx.doi.org/10.1016/j.cub.2010.09.012}, doi = {http://dx.doi.org/10.1016/j.cub.2010.09.012} }
Baker, R.P., Taormina, M.J., Jemielita, M. and Parthasarathy, R. A combined light sheet fluorescence and differential
interference contrast microscope for live imaging of
multicellular specimens
2015 J. Microsc.
Vol. 258(2), pp. 105-112 
article DOI URL 
Abstract: We describe a microscope capable of both light sheet
fluorescence microscopy and differential interference contrast
microscopy (DICM). The two imaging modes, which to the best of
our knowledge have not previously been combined, are
complementary: light sheet fluorescence microscopy provides
three-dimensional imaging of fluorescently labelled components
of multicellular systems with high speed, large fields of
view, and low phototoxicity, whereas differential interference
contrast microscopy reveals the unlabelled neighbourhood of
tissues, organs, and other structures with high contrast and
inherent optical sectioning. Use of a single Nomarski prism
for differential interference contrast microscopy and a shared
detection path for both imaging modes enables simple
integration of the two techniques in one custom microscope. We
provide several examples of the utility of the resulting
instrument, focusing especially on the digestive tract of the
larval zebrafish, revealing in this complex and heterogeneous
environment anatomical features, the behaviour of commensal
microbes, immune cell motions, and more.
BibTeX:
@article{Baker2015-qs,
  author = {Baker, R P and Taormina, M J and Jemielita, M and
Parthasarathy, R}, title = {A combined light sheet fluorescence and differential
interference contrast microscope for live imaging of
multicellular specimens}, journal = {J. Microsc.}, year = {2015}, volume = {258}, number = {2}, pages = {105--112}, url = {http://dx.doi.org/10.1111/jmi.12220}, doi = {http://dx.doi.org/10.1111/jmi.12220} }
Bassi, A., Schmid, B. and Huisken, J. Optical tomography complements light sheet microscopy for in
toto imaging of zebrafish development
2015 Development
Vol. 142(5), pp. 1016-1020 
article DOI URL 
Abstract: Fluorescently labeled structures can be spectrally isolated
and imaged at high resolution in living embryos by light sheet
microscopy. Multimodal imaging techniques are now needed to
put these distinct structures back into the context of the
surrounding tissue. We found that the bright-field contrast of
unstained specimens in a selective plane illumination
microscopy (SPIM) setup can be exploited for in vivo
tomographic reconstructions of the three-dimensional anatomy
of zebrafish, without causing phototoxicity. We report
multimodal imaging of entire zebrafish embryos over several
hours of development, as well as segmentation, tracking and
automatic registration of individual organs.
BibTeX:
@article{Bassi2015-ww,
  author = {Bassi, Andrea and Schmid, Benjamin and Huisken, Jan},
  title = {Optical tomography complements light sheet microscopy for in
toto imaging of zebrafish development}, journal = {Development}, year = {2015}, volume = {142}, number = {5}, pages = {1016--1020}, url = {http://dx.doi.org/10.1242/dev.116970}, doi = {http://dx.doi.org/10.1242/dev.116970} }
Baumgart, E. and Kubitscheck, U. Scanned light sheet microscopy with confocal slit detection 2012 Opt. Express
Vol. 20(19), pp. 21805-21814 
article DOI URL 
Abstract: In light sheet fluorescence microscopy optical sectioning is
achieved by illuminating the sample orthogonally to the
detection pathway with a thin, focused sheet of light.
However, light scattering within the sample often deteriorates
the optical sectioning effect. Here, we demonstrate that
contrast and degree of confocality can greatly be increased by
combining scanned light sheet fluorescence excitation and
confocal slit detection. A high frame rate was achieved by
using the ``rolling shutter'' of a scientific CMOS camera as a
slit detector. Synchronizing the ``rolling shutter'' with the
scanned illumination beam results in confocal line detection.
Acquiring image data with selective plane illumination
minimizes photo-damage while simultaneously enhancing
contrast, optical sectioning and signal-to-noise ratio. Thus
the imaging principle presented here merges the benefits of
scanned light sheet microscopy and line-scanning confocal
imaging.
BibTeX:
@article{Baumgart2012-qo,
  author = {Baumgart, Eugen and Kubitscheck, Ulrich},
  title = {Scanned light sheet microscopy with confocal slit detection},
  journal = {Opt. Express},
  year = {2012},
  volume = {20},
  number = {19},
  pages = {21805--21814},
  url = {http://dx.doi.org/10.1364/OE.20.021805},
  doi = {http://dx.doi.org/10.1364/OE.20.021805}
}
Becker, K., Jährling, N., Kramer, E.R., Schnorrer, F. and Dodt, H.-U. Ultramicroscopy: 3D reconstruction of large microscopical
specimens
2008 J. Biophotonics
Vol. 1(1), pp. 36-42 
article DOI URL 
Abstract: Ultramicroscopy is a microscopical technique that allows
optical sectioning and 3D reconstruction of biological and
medical specimens. While in confocal microscopy specimen size
is limited to several hundred micrometers at best, using
ultramicroscopy even centimeter sized objects like whole mouse
embryos can be reconstructed with micrometer resolution. This
is possible by using a combination of a clearing procedure and
the principle of lightsheet illumination. We present
ultramicroscopic 3D reconstructions of whole
immunohistochemically labelled mouse embryos and adult
Drosophila, giving detailed insight into their anatomy. Its
speed and simplicity makes ultramicroscopy ideally suited for
high-throughput phenotype screening of transgenic mice and
thus will benefit the investigation of disease models.
BibTeX:
@article{Becker2008-ic,
  author = {Becker, K and Jährling, N and Kramer, E R and Schnorrer,
F and Dodt, H-U}, title = {Ultramicroscopy: 3D reconstruction of large microscopical
specimens}, journal = {J. Biophotonics}, year = {2008}, volume = {1}, number = {1}, pages = {36--42}, url = {http://dx.doi.org/10.1002/jbio.200710011}, doi = {http://dx.doi.org/10.1002/jbio.200710011} }
Bhattacharya, D., Singh, V.R., Zhi, C., So, P.T.C., Matsudaira, P. and Barbastathis, G. Three dimensional HiLo-based structured illumination for a
digital scanned laser sheet microscopy (DSLM) in thick
tissue imaging
2012 Opt. Express
Vol. 20(25), pp. 27337-27347 
article DOI URL 
Abstract: Laser sheet based microscopy has become widely accepted as an
effective active illumination method for real time
three-dimensional (3D) imaging of biological tissue samples.
The light sheet geometry, where the camera is oriented
perpendicular to the sheet itself, provides an effective
method of eliminating some of the scattered light and
minimizing the sample exposure to radiation. However, residual
background noise still remains, limiting the contrast and
visibility of potentially interesting features in the samples.
In this article, we investigate additional structuring of the
illumination for improved background rejection, and propose a
new technique, ``3D HiLo'' where we combine two HiLo images
processed from orthogonal directions to improve the condition
of the 3D reconstruction. We present a comparative study of
conventional structured illumination based demodulation
methods, namely 3Phase and HiLo with a newly implemented 3D
HiLo approach and demonstrate that the latter yields superior
signal-to-background ratio in both lateral and axial
dimensions, while simultaneously suppressing image processing
artifacts.
BibTeX:
@article{Bhattacharya2012-du,
  author = {Bhattacharya, Dipanjan and Singh, Vijay Raj and Zhi, Chen and
So, Peter T C and Matsudaira, Paul and Barbastathis, George}, title = {Three dimensional HiLo-based structured illumination for a
digital scanned laser sheet microscopy (DSLM) in thick
tissue imaging}, journal = {Opt. Express}, year = {2012}, volume = {20}, number = {25}, pages = {27337--27347}, url = {http://dx.doi.org/10.1364/OE.20.027337}, doi = {http://dx.doi.org/10.1364/OE.20.027337} }
Bouchard, M.B., Voleti, V., Mendes, C.S., Lacefield, C., Grueber, W.B., Mann, R.S., Bruno, R.M. and Hillman, E.M.C. Swept confocally-aligned planar excitation (SCAPE)
microscopy for high speed volumetric imaging of behaving
organisms
2015 Nat. Photonics
Vol. 9(2), pp. 113-119 
article DOI URL 
Abstract: We report a new 3D microscopy technique that allows volumetric
imaging of living samples at ultra-high speeds: Swept,
confocally-aligned planar excitation (SCAPE) microscopy. While
confocal and two-photon microscopy have revolutionized
biomedical research, current implementations are costly,
complex and limited in their ability to image 3D volumes at
high speeds. Light-sheet microscopy techniques using
two-objective, orthogonal illumination and detection require a
highly constrained sample geometry, and either physical sample
translation or complex synchronization of illumination and
detection planes. In contrast, SCAPE microscopy acquires
images using an angled, swept light-sheet in a
single-objective, en-face geometry. Unique confocal descanning
and image rotation optics map this moving plane onto a
stationary high-speed camera, permitting completely
translationless 3D imaging of intact samples at rates
exceeding 20 volumes per second. We demonstrate SCAPE
microscopy by imaging spontaneous neuronal firing in the
intact brain of awake behaving mice, as well as freely moving
transgenic Drosophila larvae.
BibTeX:
@article{Bouchard2015-bi,
  author = {Bouchard, Matthew B and Voleti, Venkatakaushik and Mendes,
César S and Lacefield, Clay and Grueber, Wesley B and
Mann, Richard S and Bruno, Randy M and Hillman, Elizabeth M C}, title = {Swept confocally-aligned planar excitation (SCAPE)
microscopy for high speed volumetric imaging of behaving
organisms}, journal = {Nat. Photonics}, publisher = {Nature Publishing Group}, year = {2015}, volume = {9}, number = {2}, pages = {113--119}, url = {http://dx.doi.org/10.1038/nphoton.2014.323}, doi = {http://dx.doi.org/10.1038/nphoton.2014.323} }
Bourgenot, C., Saunter, C.D., Taylor, J.M., Girkin, J.M. and Love, G.D. 3D adaptive optics in a light sheet microscope 2012 Opt. Express
Vol. 20(12), pp. 13252-13261 
article DOI URL 
Abstract: We report on a single plane illumination microscope (SPIM)
incorporating adaptive optics in the imaging arm. We show how
aberrations can occur from the sample mounting tube and
quantify the aberrations both experimentally and
computationally. A wavefront sensorless approach was taken to
imaging a green fluorescent protein (GFP) labelled transgenic
zebrafish. We show improvements in image quality whilst
recording a 3D ``z-stack'' and show how the aberrations come
from varying depths in the fish.
BibTeX:
@article{Bourgenot2012-fw,
  author = {Bourgenot, Cyril and Saunter, Christopher D and Taylor,
Jonathan M and Girkin, John M and Love, Gordon D}, title = {3D adaptive optics in a light sheet microscope}, journal = {Opt. Express}, year = {2012}, volume = {20}, number = {12}, pages = {13252--13261}, url = {http://dx.doi.org/10.1364/OE.20.013252}, doi = {http://dx.doi.org/10.1364/OE.20.013252} }
Bourgenot, C., Taylor, J.M., Saunter, C.D., Girkin, J.M. and Love, G.D. Light sheet adaptive optics microscope for 3D live imaging 2013
Vol. 8589SPIE BiOS, pp. 85890W-85890W-9 
inproceedings DOI URL 
Abstract: abstract We report on the incorporation of adaptive optics (AO)
into the imaging arm of a selective plane illumination
microscope (SPIM). SPIM has recently emerged as an important
tool for life science research due to its ability to deliver
high-speed, optically sectioned, ...
BibTeX:
@inproceedings{Bourgenot2013-dm,
  author = {Bourgenot, C and Taylor, J M and Saunter, C D and Girkin, J M
and Love, G D}, title = {Light sheet adaptive optics microscope for 3D live imaging}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2013}, volume = {8589}, pages = {85890W--85890W--9}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1656298}, doi = {http://dx.doi.org/10.1117/12.2002057} }
Bradl, J., Hausmann, M., Ehemann, V., Komitowski, D. and Cremer, C. A tilting device for three-dimensional microscopy: application
to in situ imaging of interphase cell nuclei
1992 J. Microsc.
Vol. 168(Pt 1), pp. 47-57 
article URL 
Abstract: The resolution of an optical microscope is considerably less
in the direction of the optical axis (z) than in the focal
plane (x-y plane). This is true of conventional as well as
confocal microscopes. For quantitative microscopy, for
instance studies of the three-dimensional (3-D) organization
of chromosomes in human interphase cell nuclei, the 3-D image
must be reconstructed by a point spread function or an optical
transfer function with careful consideration of the properties
of the imaging system. To alleviate the reconstruction
problem, a tilting device was developed so that several data
sets of the same cell nucleus under different views could be
registered. The 3-D information was obtained from a series of
optical sections with a Zeiss transmission light microscope
Axiomat using a stage with a computer-controlled stepping
motor for movement in the z-axis. The tilting device on the
Axiomat stage could turn a cell nucleus through any desired
angle and also provide movement in the x-y direction. The
technique was applied to 3-D imaging of human lymphocyte cell
nuclei, which were labelled by in situ hybridization with the
DNA probe pUC 1.77 (mainly specific for chromosome 1). For
each nucleus, 3-D data sets were registered at viewing angles
of 0 degrees, 90 degrees and 180 degrees; the volumes and
positions of the labelled regions (spots) were calculated. The
results also confirm that, in principle, any angle of a 2 pi
geometry can be fixed for data acquisition with a high
reproducibility. This indicates the feasibility of
axiotomographical microscopy of cell nuclei.
BibTeX:
@article{Bradl1992-ie,
  author = {Bradl, J and Hausmann, M and Ehemann, V and Komitowski, D and
Cremer, C}, title = {A tilting device for three-dimensional microscopy: application
to in situ imaging of interphase cell nuclei}, journal = {J. Microsc.}, year = {1992}, volume = {168}, number = {Pt 1}, pages = {47--57}, url = {http://www.ncbi.nlm.nih.gov/pubmed/1447754} }
Brede, C., Friedrich, M., Jordán-Garrote, A.-L., Riedel, S.S., Bäuerlein, C.A., Heinze, K.G., Bopp, T., Schulz, S., Mottok, A., Kiesel, C., Mattenheimer, K., Ritz, M., von Krosigk, V., Rosenwald, A., Einsele, H., Negrin, R.S., Harms, G.S. and Beilhack, A. Mapping immune processes in intact tissues at cellular
resolution
2012 J. Clin. Invest.
Vol. 122(12), pp. 4439-4446 
article DOI URL 
Abstract: Understanding the spatiotemporal changes of cellular and
molecular events within an organism is crucial to elucidate
the complex immune processes involved in infections,
autoimmune disorders, transplantation, and neoplastic
transformation and metastasis. Here we introduce a novel
multicolor light sheet fluorescence microscopy (LSFM) approach
for deciphering immune processes in large tissue specimens on
a single-cell level in 3 dimensions. We combined and optimized
antibody penetration, tissue clearing, and triple-color
illumination to create a method for analyzing intact mouse and
human tissues. This approach allowed us to successfully
quantify changes in expression patterns of mucosal vascular
addressin cell adhesion molecule-1 (MAdCAM-1) and T cell
responses in Peyer's patches following stimulation of the
immune system. In addition, we employed LSFM to map individual
T cell subsets after hematopoietic cell transplantation and
detected rare cellular events. Thus, we present a versatile
imaging technology that should be highly beneficial in
biomedical research.
BibTeX:
@article{Brede2012-kx,
  author = {Brede, Christian and Friedrich, Mike and Jordán-Garrote,
Ana-Laura and Riedel, Simone S and Bäuerlein, Carina A
and Heinze, Katrin G and Bopp, Tobias and Schulz, Stephan and
Mottok, Anja and Kiesel, Carolin and Mattenheimer, Katharina
and Ritz, Miriam and von Krosigk, Viktoria and Rosenwald,
Andreas and Einsele, Hermann and Negrin, Robert S and Harms,
Gregory S and Beilhack, Andreas}, title = {Mapping immune processes in intact tissues at cellular
resolution}, journal = {J. Clin. Invest.}, year = {2012}, volume = {122}, number = {12}, pages = {4439--4446}, url = {http://dx.doi.org/10.1172/JCI65100}, doi = {http://dx.doi.org/10.1172/JCI65100} }
Breuninger, T., Greger, K. and Stelzer, E.H.K. Lateral modulation boosts image quality in single plane
illumination fluorescence microscopy
2007 Opt. Lett.
Vol. 32(13), pp. 1938-1940 
article URL 
Abstract: A new microscope combines optical sectioning by fluorophore
excitation using a single light sheet with structured
illumination. Several images with laterally
intensity-modulated light sheets are recorded from scattering
fluorescent specimens. By applying a simple data processing
scheme, the nonmodulated volumes are identified. The blurred
features become dark, and the resultant images are improved in
terms of contrast and resolution. Hence, the instrument is
capable of discriminating against contributions to the image
that are induced by the optical properties of the specimen.
The new microscope's capabilities are demonstrated by imaging
the internals of the head of an adult Drosophila melanogaster
(fruit fly) expressing green fluorescent protein-labeled
polycomb proteins.
BibTeX:
@article{Breuninger2007-jp,
  author = {Breuninger, Tobias and Greger, Klaus and Stelzer, Ernst H K},
  title = {Lateral modulation boosts image quality in single plane
illumination fluorescence microscopy}, journal = {Opt. Lett.}, year = {2007}, volume = {32}, number = {13}, pages = {1938--1940}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17603620} }
Bruns, T., Schickinger, S. and Schneckenburger, H. Sample holder for axial rotation of specimens in 3D
microscopy
2015 J. Microsc.
Vol. 260(1), pp. 30-36 
article DOI URL 
Abstract: In common light microscopy, observation of samples is only
possible from one perspective. However, especially for larger
three-dimensional specimens observation from different views
is desirable. Therefore, we are presenting a sample holder
permitting rotation of the specimen around an axis
perpendicular to the light path of the microscope. Thus,
images can be put into a defined multidimensional context,
enabling reliable three-dimensional reconstructions. The
device can be easily adapted to a great variety of common
light microscopes and is suitable for various applications in
science, education and industry, where the observation of
three-dimensional specimens is essential. Fluorescence
z-projection images of copepods and ixodidae ticks at
different rotation angles obtained by confocal laser scanning
microscopy and light sheet fluorescence microscopy are
reported as representative results.
BibTeX:
@article{Bruns2015-qb,
  author = {Bruns, T and Schickinger, S and Schneckenburger, H},
  title = {Sample holder for axial rotation of specimens in 3D
microscopy}, journal = {J. Microsc.}, year = {2015}, volume = {260}, number = {1}, pages = {30--36}, url = {http://dx.doi.org/10.1111/jmi.12263}, doi = {http://dx.doi.org/10.1111/jmi.12263} }
Bruns, T., Schickinger, S. and Schneckenburger, H. Single plane illumination module and micro-capillary approach
for a wide-field microscope
2014 J. Vis. Exp.(90), pp. e51993  article DOI URL 
Abstract: A module for light sheet or single plane illumination
microscopy (SPIM) is described which is easily adapted to an
inverted wide-field microscope and optimized for 3-dimensional
cell cultures, e.g., multi-cellular tumor spheroids (MCTS).
The SPIM excitation module shapes and deflects the light such
that the sample is illuminated by a light sheet perpendicular
to the detection path of the microscope. The system is
characterized by use of a rectangular capillary for holding
(and in an advanced version also by a micro-capillary approach
for rotating) the samples, by synchronous adjustment of the
illuminating light sheet and the objective lens used for
fluorescence detection as well as by adaptation of a
microfluidic system for application of fluorescent dyes,
pharmaceutical agents or drugs in small quantities. A protocol
for working with this system is given, and some technical
details are reported. Representative results include (1)
measurements of the uptake of a cytostatic drug (doxorubicin)
and its partial conversion to a degradation product, (2) redox
measurements by use of a genetically encoded glutathione
sensor upon addition of an oxidizing agent, and (3) initiation
and labeling of cell necrosis upon inhibition of the
mitochondrial respiratory chain. Differences and advantages of
the present SPIM module in comparison with existing systems
are discussed.
BibTeX:
@article{Bruns2014-lh,
  author = {Bruns, Thomas and Schickinger, Sarah and Schneckenburger,
Herbert}, title = {Single plane illumination module and micro-capillary approach
for a wide-field microscope}, journal = {J. Vis. Exp.}, year = {2014}, number = {90}, pages = {e51993}, url = {http://dx.doi.org/10.3791/51993}, doi = {http://dx.doi.org/10.3791/51993} }
Bruns, T., Schickinger, S., Wittig, R. and Schneckenburger, H. Preparation strategy and illumination of three-dimensional
cell cultures in light sheet-based fluorescence microscopy
2012 J. Biomed. Opt.
Vol. 17(10), pp. 101518 
article DOI URL 
Abstract: A device for selective plane illumination microscopy (SPIM) of
three-dimensional multicellular spheroids, in culture medium
under stationary or microfluidic conditions, is described.
Cell spheroids are located in a micro-capillary and a light
sheet, for illumination, is generated in an optical setup
adapted to a conventional inverse microscope. Layers of the
sample, of about 10 $m or less in diameter, are, thus,
illuminated selectively and imaged by high resolution
fluorescence microscopy. SPIM is operated at low light
exposure even if a larger number of layers is imaged and is
easily combined with laser scanning microscopy. Chinese
hamster ovary cells expressing a membrane-associated green
fluorescent protein are used for preliminary tests, and the
uptake of the fluorescent marker, acridine orange via a
microfluidic system, is visualized to demonstrate its
potential in cancer research such as for the detection of
cellular responses to anticancer drugs.
BibTeX:
@article{Bruns2012-fe,
  author = {Bruns, Thomas and Schickinger, Sarah and Wittig, Rainer and
Schneckenburger, Herbert}, title = {Preparation strategy and illumination of three-dimensional
cell cultures in light sheet-based fluorescence microscopy}, journal = {J. Biomed. Opt.}, publisher = {International Society for Optics and Photonics}, year = {2012}, volume = {17}, number = {10}, pages = {101518}, url = {http://dx.doi.org/10.1117/1.JBO.17.10.101518}, doi = {http://dx.doi.org/10.1117/1.JBO.17.10.101518} }
Buytaert, J.A.N., Descamps, E., Adriaens, D. and Dirckx, J.J.J. The OPFOS Microscopy Family: High-Resolution Optical
Sectioning of Biomedical Specimens
2012 Anat. Res. Int.
Vol. 2012, pp. 206238 
article DOI URL 
Abstract: We report on the recently emerging (laser) light-sheet-based
fluorescence microscopy field (LSFM). The techniques used in
this field allow to study and visualize biomedical objects
nondestructively in high resolution through virtual optical
sectioning with sheets of laser light. Fluorescence
originating in the cross-section of the sheet and sample is
recorded orthogonally with a camera. In this paper, the first
implementation of LSFM to image biomedical tissue in three
dimensions-orthogonal-plane fluorescence optical sectioning
microscopy (OPFOS)-is discussed. Since then many similar and
derived methods have surfaced, (SPIM, ultramicroscopy,
HR-OPFOS, mSPIM, DSLM, TSLIM, etc.) which we all briefly
discuss. All these optical sectioning methods create images
showing histological detail. We illustrate the applicability
of LSFM on several specimen types with application in
biomedical and life sciences.
BibTeX:
@article{Buytaert2012-fu,
  author = {Buytaert, Jan A N and Descamps, Emilie and Adriaens, Dominique
and Dirckx, Joris J J}, title = {The OPFOS Microscopy Family: High-Resolution Optical
Sectioning of Biomedical Specimens}, journal = {Anat. Res. Int.}, year = {2012}, volume = {2012}, pages = {206238}, url = {http://dx.doi.org/10.1155/2012/206238}, doi = {http://dx.doi.org/10.1155/2012/206238} }
Buytaert, J.A.N. and Dirckx, J.J.J. Tomographic imaging of macroscopic biomedical objects in high
resolution and three dimensions using orthogonal-plane
fluorescence optical sectioning
2009 Appl. Opt.
Vol. 48(5), pp. 941-948 
article URL 
Abstract: A new optical-fluorescence microscopy technique, called
HR-OPFOS, is discussed and situated among similar
OPFOS-implementations. OPFOS stands for orthogonal-plane
fluorescence optical sectioning and thus is categorized as a
laser light sheet based fluorescence microscopy method.
HR-OPFOS is used to make tomographic recordings of macroscopic
biomedical specimens in high resolution. It delivers cross
sections through the object under study with semi-histological
detail, which can be used to create three-dimensional computer
models for finite-element modeling or anatomical studies. The
general innovation of this class of microscopy setup consists
of the separation of the illumination and observation axes,
but now in our setup combined with focal line scanning to
improve sectioning resolution. HR-OPFOS is demonstrated on
gerbil hearing organs and on mouse and bird brains. The
necessary specimen preparation is discussed.
BibTeX:
@article{Buytaert2009-xk,
  author = {Buytaert, Jan A N and Dirckx, Joris J J},
  title = {Tomographic imaging of macroscopic biomedical objects in high
resolution and three dimensions using orthogonal-plane
fluorescence optical sectioning}, journal = {Appl. Opt.}, year = {2009}, volume = {48}, number = {5}, pages = {941--948}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19209207} }
Buytaert, J.A.N. and Dirckx, J.J.J. Design and quantitative resolution measurements of an optical
virtual sectioning three-dimensional imaging technique for
biomedical specimens, featuring two-micrometer slicing
resolution
2007 J. Biomed. Opt.
Vol. 12(1), pp. 014039 
article DOI URL 
Abstract: Several well-established techniques are available to obtain
3-D image information of biomedical specimens, each with their
specific advantages and limitations. Orthogonal plane
fluorescence optical sectioning (OPFOS), or selective plane
illumination microscopy (SPIM), are additional techniques
which, after adequate specimen preparation, produce high
quality, autoaligned sectional images in nearly real time, of
bone as well as soft tissue. Up until now, slicing resolutions
down to 14 microm have been obtained. We present a high
resolution (HR) OPFOS method, which delivers images that
approach the quality of histological sections. With our
HROPFOS technique, we achieve in-plane resolutions of 1 microm
and a slicing resolution of 2 microm. A region of interest
within an intact and much larger object can be imaged without
problems, and as the optical technique is nondestructive, the
object can be measured in any slicing direction. We present
quantitative measurements of resolution. A 3-D model
reconstructed from our HROPFOS data is compared to SEM
results, and the technique is demonstrated with section images
and 3-D reconstructions of middle ear specimens.
BibTeX:
@article{Buytaert2007-jb,
  author = {Buytaert, Jan A N and Dirckx, Joris J J},
  title = {Design and quantitative resolution measurements of an optical
virtual sectioning three-dimensional imaging technique for
biomedical specimens, featuring two-micrometer slicing
resolution}, journal = {J. Biomed. Opt.}, year = {2007}, volume = {12}, number = {1}, pages = {014039}, url = {http://dx.doi.org/10.1117/1.2671712}, doi = {http://dx.doi.org/10.1117/1.2671712} }
Can, A., Al-Kofahi, O., Lasek, S., Szarowski, D.H., Turner, J.N. and Roysam, B. Attenuation correction in confocal laser microscopes: a novel
two-view approach
2003 J. Microsc.
Vol. 211(Pt 1), pp. 67-79 
article URL 
Abstract: Confocal microscopy is a three-dimensional (3D) imaging
modality, but the specimen thickness that can be imaged is
limited by depth-dependent signal attenuation. Both software
and hardware methods have been used to correct the attenuation
in reconstructed images, but previous methods do not increase
the image signal-to-noise ratio (SNR) using conventional
specimen preparation and imaging. We present a practical
two-view method that increases the overall imaging depth,
corrects signal attenuation and improves the SNR. This is
achieved by a combination of slightly modified but
conventional specimen preparation, image registration, montage
synthesis and signal reconstruction methods. The specimen is
mounted in a symmetrical manner between a pair of cover slips,
rather than between a slide and a cover slip. It is imaged
sequentially from both sides to generate two 3D image stacks
from perspectives separated by approximately 180 degrees with
respect to the optical axis. An automated image registration
algorithm performs a precise 3D alignment, and a model-based
minimum mean squared algorithm synthesizes a montage,
combining the content of both the 3D views. Experiments with
images of individual neurones contrasted with a space-filling
fluorescent dye in thick brain tissue slices produced precise
3D montages that are corrected for depth-dependent signal
attenuation. The SNR of the reconstructed image is maximized
by the method, and it is significantly higher than in the
single views after applying our attenuation model. We also
compare our method with simpler two-view reconstruction
methods and quantify the SNR improvement. The reconstructed
images are a more faithful qualitative visualization of the
specimen's structure and are quantitatively more accurate,
providing a more rigorous basis for automated image analysis.
BibTeX:
@article{Can2003-vk,
  author = {Can, A and Al-Kofahi, O and Lasek, S and Szarowski, D H and
Turner, J N and Roysam, B}, title = {Attenuation correction in confocal laser microscopes: a novel
two-view approach}, journal = {J. Microsc.}, year = {2003}, volume = {211}, number = {Pt 1}, pages = {67--79}, url = {http://www.ncbi.nlm.nih.gov/pubmed/12839553} }
Capoulade, J., Reynaud, E.G. and Wachsmuth, M. Imaging Marine Life with a Thin Light-Sheet 2013 Imaging Marine Life, pp. 186-209  incollection DOI URL 
Abstract: Light-sheet fluorescence microscopy (LSFM) is a non-invasive
optical method for the observation of living specimens. Although
this concept was established a century ago it is only during the
last decade that instruments suitable for biological
applications have been developed, thereby circumventing some of
the limitations of established fluorescence imaging techniques
such as confocal laser scanning microscopy. LSFM utilizes a
sheet of laser light to illuminate only a thin slice of a
fluorescently labeled sample. A wide-field fluorescence
microscope, placed perpendicular to the light-sheet, serves to
collect the fluorescence signal and image the observed region by
means of a camera. This side-on illumination configuration
features several advantages, including intrinsic optical
sectioning without the need of spatial filtering as employed in
confocal microscopy, excellent signal-to-noise ratio, high
temporal resolution, and drastically reduced overall
photobleaching and phototoxicity inside living specimens.
Moreover, the non-conventional geometry of LSFM opens up a
completely new way of sample mounting, enabling convenient
multi-view image acquisition for 3D imaging by simple rotation
of the sample within the medium-filled chamber. Although LSFM
was developed originally for the observation of large organisms
such as zebrafish embryos, this method can be adapted to a large
range of samples from macroscopic specimens like corals or
copepods to microscopic organisms like tintinnids.
BibTeX:
@incollection{Capoulade2013-xi,
  author = {Capoulade, Jérémie and Reynaud, Emmanuel G and
Wachsmuth, Malte}, title = {Imaging Marine Life with a Thin Light-Sheet}, booktitle = {Imaging Marine Life}, publisher = {Wiley-VCH Verlag GmbH & Co. KGaA}, year = {2013}, pages = {186--209}, url = {http://dx.doi.org/10.1002/9783527675418.ch8}, doi = {http://dx.doi.org/10.1002/9783527675418.ch8} }
Capoulade, J., Wachsmuth, M., Hufnagel, L. and Knop, M. Quantitative fluorescence imaging of protein diffusion and
interaction in living cells
2011 Nat. Biotechnol.
Vol. 29(9), pp. 835-839 
article DOI URL 
Abstract: Diffusion processes and local dynamic equilibria inside cells
lead to nonuniform spatial distributions of molecules, which
are essential for processes such as nuclear organization and
signaling in cell division, differentiation and migration. To
understand these mechanisms, spatially resolved quantitative
measurements of protein abundance, mobilities and interactions
are needed, but current methods have limited capabilities to
study dynamic parameters. Here we describe a microscope based
on light-sheet illumination that allows massively parallel
fluorescence correlation spectroscopy (FCS) measurements and
use it to visualize the diffusion and interactions of proteins
in mammalian cells and in isolated fly tissue. Imaging the
mobility of heterochromatin protein HP1$ (ref. 4) in
cell nuclei we could provide high-resolution diffusion maps
that reveal euchromatin areas with heterochromatin-like
HP1$-chromatin interactions. We expect that FCS imaging
will become a useful method for the precise characterization
of cellular reaction-diffusion processes.
BibTeX:
@article{Capoulade2011-jd,
  author = {Capoulade, Jérémie and Wachsmuth, Malte and Hufnagel,
Lars and Knop, Michael}, title = {Quantitative fluorescence imaging of protein diffusion and
interaction in living cells}, journal = {Nat. Biotechnol.}, year = {2011}, volume = {29}, number = {9}, pages = {835--839}, url = {http://dx.doi.org/10.1038/nbt.1928}, doi = {http://dx.doi.org/10.1038/nbt.1928} }
Cavey, M. and Lecuit, T. Imaging cellular and molecular dynamics in live embryos using
fluorescent proteins
2008 Methods Mol. Biol.
Vol. 420, pp. 219-238 
article DOI URL 
Abstract: With the live imaging of embryos, the dynamics of
developmental processes, such as tissue remodeling, cell
morphogenesis, and, campus protein dynamics can be observed
and quantified. This has greatly improved the mechanistic
understanding of biological processes. Here we describe how
embryos can be prepared for imaging mainly, but not only,
fluorescent proteins and probes. This chapter is a users'
guide that addresses the following aspects of fluorescent
embryo imaging: (1) How to handle and prepare embryos for live
microscopy. (2) What microscopic setups are available for
embryo imaging and what should they be used for. (3) How to
practically use fluorescent imaging setups depending on the
experimental context: large-scale imaging of multiple embryos,
high-resolution four-dimensional imaging of single embryos,
studies of protein dynamics, and so on. (4) Finally, we focus
on pitfalls and how to overcome a variety of possible problems
encountered during live imaging.
BibTeX:
@article{Cavey2008-au,
  author = {Cavey, Matthieu and Lecuit, Thomas},
  title = {Imaging cellular and molecular dynamics in live embryos using
fluorescent proteins}, journal = {Methods Mol. Biol.}, year = {2008}, volume = {420}, pages = {219--238}, url = {http://dx.doi.org/10.1007/978-1-59745-583-1_13}, doi = {http://dx.doi.org/10.1007/978-1-59745-583-1\_13} }
Cella Zanacchi, F., Lavagnino, Z., Faretta, M., Furia, L. and Diaspro, A. Light-sheet confined super-resolution using two-photon
photoactivation
2013 PLoS One
Vol. 8(7), pp. e67667 
article DOI URL 
Abstract: Light-sheet microscopy is a useful tool for performing
biological investigations of thick samples and it has recently
been demonstrated that it can also act as a suitable
architecture for super-resolution imaging of thick biological
samples by means of individual molecule localization. However,
imaging in depth is still limited since it suffers from a
reduction in image quality caused by scattering effects. This
paper sets out to investigate the advantages of non-linear
photoactivation implemented in a selective plane illumination
configuration when imaging scattering samples. In particular,
two-photon excitation is proven to improve imaging
capabilities in terms of imaging depth and is expected to
reduce light-sample interactions and sample photo-damage.
Here, two-photon photoactivation is coupled to individual
molecule localization methods based on light-sheet
illumination (IML-SPIM), allowing super-resolution imaging of
nuclear pH2AX in NB4 cells.
BibTeX:
@article{Cella_Zanacchi2013-wy,
  author = {Cella Zanacchi, Francesca and Lavagnino, Zeno and Faretta,
Mario and Furia, Laura and Diaspro, Alberto}, title = {Light-sheet confined super-resolution using two-photon
photoactivation}, journal = {PLoS One}, year = {2013}, volume = {8}, number = {7}, pages = {e67667}, url = {http://dx.doi.org/10.1371/journal.pone.0067667}, doi = {http://dx.doi.org/10.1371/journal.pone.0067667} }
Cella Zanacchi, F., Lavagnino, Z., Perrone Donnorso, M., Del Bue, A., Furia, L., Faretta, M. and Diaspro, A. Live-cell 3D super-resolution imaging in thick biological
samples
2011 Nat. Methods
Vol. 8(12), pp. 1047-1049 
article DOI URL 
Abstract: We demonstrate three-dimensional (3D) super-resolution
live-cell imaging through thick specimens (50-150 $m), by
coupling far-field individual molecule localization with
selective plane illumination microscopy (SPIM). The improved
signal-to-noise ratio of selective plane illumination allows
nanometric localization of single molecules in thick
scattering specimens without activating or exciting molecules
outside the focal plane. We report 3D super-resolution imaging
of cellular spheroids.
BibTeX:
@article{Cella_Zanacchi2011-nd,
  author = {Cella Zanacchi, Francesca and Lavagnino, Zeno and Perrone
Donnorso, Michela and Del Bue, Alessio and Furia, Laura and
Faretta, Mario and Diaspro, Alberto}, title = {Live-cell 3D super-resolution imaging in thick biological
samples}, journal = {Nat. Methods}, year = {2011}, volume = {8}, number = {12}, pages = {1047--1049}, url = {http://dx.doi.org/10.1038/nmeth.1744}, doi = {http://dx.doi.org/10.1038/nmeth.1744} }
Cella Zanacchi, F., Lavagnino, Z., Pesce, M., Difato, F., Ronzitti, E. and Diaspro, A. Two-photon fluorescence excitation within a light sheet based
microscopy architecture
2011 SPIE BiOS, pp. 79032W-79032W-5  inproceedings DOI URL 
BibTeX:
@inproceedings{Cella_Zanacchi2011-rx,
  author = {Cella Zanacchi, F and Lavagnino, Z and Pesce, M and Difato, F
and Ronzitti, E and Diaspro, A}, title = {Two-photon fluorescence excitation within a light sheet based
microscopy architecture}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2011}, pages = {79032W--79032W--5}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=719074}, doi = {http://dx.doi.org/10.1117/12.879792} }
Chacko, N., Chan, K.G. and Liebling, M. Intensity-Based Point-Spread-Function-Aware Registration for
Multi-View Applications in Optical Microscopy
2015 Biomedical Imaging (ISBI), 2015 IEEE 12th International
Symposium on 
inproceedings URL 
BibTeX:
@inproceedings{Chacko2015-cg,
  author = {Chacko, Nikhil and Chan, Kevin G and Liebling, Michael},
  title = {Intensity-Based Point-Spread-Function-Aware Registration for
Multi-View Applications in Optical Microscopy}, booktitle = {Biomedical Imaging (ISBI), 2015 IEEE 12th International
Symposium on}, year = {2015}, url = {http://publications.idiap.ch/downloads/papers/2015/Chacko_ISBI15_2015.pdf} }
Chan, K.G. and Liebling, M. Estimation of Divergence-Free 3D Cardiac Blood Flow in a
Zebrafish Larva Using Multi-View Microscopy
2015 IEEE-Biomedical Imaging (ISBI), 2015 IEEE 12th
International Symposium on 
inproceedings URL 
BibTeX:
@inproceedings{Chan2015-jt,
  author = {Chan, Kevin G and Liebling, Michael},
  title = {Estimation of Divergence-Free 3D Cardiac Blood Flow in a
Zebrafish Larva Using Multi-View Microscopy}, booktitle = {IEEE-Biomedical Imaging (ISBI), 2015 IEEE 12th
International Symposium on}, year = {2015}, url = {http://publications.idiap.ch/downloads/papers/2015/Chan_ISBI_2015.pdf} }
Chardès, C., Mélénec, P., Bertrand, V. and Lenne, P.-F. Setting up a simple light sheet microscope for in toto imaging
of C. elegans development
2014 J. Vis. Exp.(87)  article DOI URL 
Abstract: Fast and low phototoxic imaging techniques are pre-requisite
to study the development of organisms in toto. Light sheet
based microscopy reduces photo-bleaching and phototoxic
effects compared to confocal microscopy, while providing 3D
images with subcellular resolution. Here we present the setup
of a light sheet based microscope, which is composed of an
upright microscope and a small set of opto-mechanical elements
for the generation of the light sheet. The protocol describes
how to build, align the microscope and characterize the light
sheet. In addition, it details how to implement the method for
in toto imaging of C. elegans embryos using a simple
observation chamber. The method allows the capture of 3D
two-colors time-lapse movies over few hours of development.
This should ease the tracking of cell shape, cell divisions
and tagged proteins over long periods of time.
BibTeX:
@article{Chardes2014-gq,
  author = {Chardès, Claire and Mélénec, Pauline and Bertrand,
Vincent and Lenne, Pierre-François}, title = {Setting up a simple light sheet microscope for in toto imaging
of C. elegans development}, journal = {J. Vis. Exp.}, year = {2014}, number = {87}, url = {http://dx.doi.org/10.3791/51342}, doi = {http://dx.doi.org/10.3791/51342} }
Chen, B.-C., Legant, W.R., Wang, K., Shao, L., Milkie, D.E., Davidson, M.W., Janetopoulos, C., Wu, X.S., Hammer 3rd, J.A., Liu, Z., English, B.P., Mimori-Kiyosue, Y., Romero, D.P., Ritter, A.T., Lippincott-Schwartz, J., Fritz-Laylin, L., Mullins, R.D., Mitchell, D.M., Bembenek, J.N., Reymann, A.-C., Böhme, R., Grill, S.W., Wang, J.T., Seydoux, G., Tulu, U.S., Kiehart, D.P. and Betzig, E. Lattice light-sheet microscopy: imaging molecules to embryos
at high spatiotemporal resolution
2014 Science
Vol. 346(6208), pp. 1257998 
article DOI URL 
Abstract: Although fluorescence microscopy provides a crucial window
into the physiology of living specimens, many biological
processes are too fragile, are too small, or occur too rapidly
to see clearly with existing tools. We crafted ultrathin light
sheets from two-dimensional optical lattices that allowed us
to image three-dimensional (3D) dynamics for hundreds of
volumes, often at subsecond intervals, at the diffraction
limit and beyond. We applied this to systems spanning four
orders of magnitude in space and time, including the diffusion
of single transcription factor molecules in stem cell
spheroids, the dynamic instability of mitotic microtubules,
the immunological synapse, neutrophil motility in a 3D matrix,
and embryogenesis in Caenorhabditis elegans and Drosophila
melanogaster. The results provide a visceral reminder of the
beauty and the complexity of living systems.
BibTeX:
@article{Chen2014-qu,
  author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao,
Lin and Milkie, Daniel E and Davidson, Michael W and
Janetopoulos, Chris and Wu, Xufeng S and Hammer, 3rd, John A
and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and
Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz,
Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and
Mitchell, Diana M and Bembenek, Joshua N and Reymann,
Anne-Cecile and Böhme, Ralph and Grill, Stephan W and
Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and
Kiehart, Daniel P and Betzig, Eric}, title = {Lattice light-sheet microscopy: imaging molecules to embryos
at high spatiotemporal resolution}, journal = {Science}, year = {2014}, volume = {346}, number = {6208}, pages = {1257998}, url = {http://dx.doi.org/10.1126/science.1257998}, doi = {http://dx.doi.org/10.1126/science.1257998} }
Chi, N.C., Shaw, R.M., Jungblut, B., Huisken, J., Ferrer, T., Arnaout, R., Scott, I., Beis, D., Xiao, T., Baier, H., Jan, L.Y., Tristani-Firouzi, M. and Stainier, D.Y.R. Genetic and physiologic dissection of the vertebrate cardiac
conduction system
2008 PLoS Biol.
Vol. 6(5), pp. e109 
article DOI URL 
Abstract: Vertebrate hearts depend on highly specialized cardiomyocytes
that form the cardiac conduction system (CCS) to coordinate
chamber contraction and drive blood efficiently and
unidirectionally throughout the organism. Defects in this
specialized wiring system can lead to syncope and sudden
cardiac death. Thus, a greater understanding of cardiac
conduction development may help to prevent these devastating
clinical outcomes. Utilizing a cardiac-specific fluorescent
calcium indicator zebrafish transgenic line,
Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping
analysis in intact animals, we identified and analyzed four
distinct stages of cardiac conduction development that
correspond to cellular and anatomical changes of the
developing heart. Additionally, we observed that epigenetic
factors, such as hemodynamic flow and contraction, regulate
the fast conduction network of this specialized electrical
system. To identify novel regulators of the CCS, we designed
and performed a new, physiology-based, forward genetic screen
and identified for the first time, to our knowledge, 17
conduction-specific mutations. Positional cloning of
hobgoblin(s634) revealed that tcf2, a homeobox transcription
factor gene involved in mature onset diabetes of the young and
familial glomerulocystic kidney disease, also regulates
conduction between the atrium and the ventricle. The
combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical
mapping technique and characterization of cardiac conduction
mutants provides a novel multidisciplinary approach to further
understand the molecular determinants of the vertebrate CCS.
BibTeX:
@article{Chi2008-hn,
  author = {Chi, Neil C and Shaw, Robin M and Jungblut, Benno and Huisken,
Jan and Ferrer, Tania and Arnaout, Rima and Scott, Ian and
Beis, Dimitris and Xiao, Tong and Baier, Herwig and Jan, Lily
Y and Tristani-Firouzi, Martin and Stainier, Didier Y R}, title = {Genetic and physiologic dissection of the vertebrate cardiac
conduction system}, journal = {PLoS Biol.}, year = {2008}, volume = {6}, number = {5}, pages = {e109}, url = {http://dx.doi.org/10.1371/journal.pbio.0060109}, doi = {http://dx.doi.org/10.1371/journal.pbio.0060109} }
Chittajallu, D.R., Florian, S., Kohler, R.H., Iwamoto, Y., Orth, J.D., Weissleder, R., Danuser, G. and Mitchison, T.J. In vivo cell-cycle profiling in xenograft tumors by
quantitative intravital microscopy
2015 Nat. Methods
Vol. 12(6), pp. 577-585 
article DOI URL 
Abstract: Quantification of cell-cycle state at a single-cell level is
essential to understand fundamental three-dimensional (3D)
biological processes such as tissue development and cancer.
Analysis of 3D in vivo images, however, is very challenging.
Today's best practice, manual annotation of select image
events, generates arbitrarily sampled data distributions,
which are unsuitable for reliable mechanistic inferences.
Here, we present an integrated workflow for quantitative in
vivo cell-cycle profiling. It combines image analysis and
machine learning methods for automated 3D segmentation and
cell-cycle state identification of individual cell-nuclei with
widely varying morphologies embedded in complex tumor
environments. We applied our workflow to quantify cell-cycle
effects of three antimitotic cancer drugs over 8 d in HT-1080
fibrosarcoma xenografts in living mice using a data set of
38,000 cells and compared the induced phenotypes. In contrast
to results with 2D culture, observed mitotic arrest was
relatively low, suggesting involvement of additional
mechanisms in their antitumor effect in vivo.
BibTeX:
@article{Chittajallu2015-ji,
  author = {Chittajallu, Deepak R and Florian, Stefan and Kohler, Rainer H
and Iwamoto, Yoshiko and Orth, James D and Weissleder, Ralph
and Danuser, Gaudenz and Mitchison, Timothy J}, title = {In vivo cell-cycle profiling in xenograft tumors by
quantitative intravital microscopy}, journal = {Nat. Methods}, year = {2015}, volume = {12}, number = {6}, pages = {577--585}, url = {http://dx.doi.org/10.1038/nmeth.3363}, doi = {http://dx.doi.org/10.1038/nmeth.3363} }
Chmielewski, A.K., Kyrsting, A., Mahou, P., Wayland, M.T., Muresan, L., Evers, J.F. and Kaminski, C.F. Fast imaging of live organisms with sculpted light sheets 2015 Sci. Rep.
Vol. 5, pp. 9385 
article DOI URL 
Abstract: Light-sheet microscopy is an increasingly popular technique in
the life sciences due to its fast 3D imaging capability of
fluorescent samples with low photo toxicity compared to
confocal methods. In this work we present a new, fast,
flexible and simple to implement method to optimize the
illumination light-sheet to the requirement at hand. A
telescope composed of two electrically tuneable lenses enables
us to define thickness and position of the light-sheet
independently but accurately within milliseconds, and
therefore optimize image quality of the features of interest
interactively. We demonstrated the practical benefit of this
technique by 1) assembling large field of views from tiled
single exposure each with individually optimized illumination
settings; 2) sculpting the light-sheet to trace complex sample
shapes within single exposures. This technique proved
compatible with confocal line scanning detection, further
improving image contrast and resolution. Finally, we
determined the effect of light-sheet optimization in the
context of scattering tissue, devising procedures for
balancing image quality, field of view and acquisition speed.
BibTeX:
@article{Chmielewski2015-ms,
  author = {Chmielewski, Aleksander K and Kyrsting, Anders and Mahou,
Pierre and Wayland, Matthew T and Muresan, Leila and Evers,
Jan Felix and Kaminski, Clemens F}, title = {Fast imaging of live organisms with sculpted light sheets}, journal = {Sci. Rep.}, year = {2015}, volume = {5}, pages = {9385}, url = {http://dx.doi.org/10.1038/srep09385}, doi = {http://dx.doi.org/10.1038/srep09385} }
Chow, C.L., Guo, W., Trivedi, P., Zhao, X. and Gubbels, S.P. Characterization of a unique cell population marked by
transgene expression in the adult cochlea of
nestin-CreER(T2)/tdTomato-reporter mice
2015 J. Comp. Neurol.
Vol. 523(10), pp. 1474-1487 
article DOI URL 
Abstract: Hair cells in the adult mammalian cochlea cannot spontaneously
regenerate after damage, resulting in the permanency of
hearing loss. Stem cells have been found to be present in the
cochlea of young rodents; however, there has been little
evidence for their existence into adulthood. We used
nestin-CreER(T2)/tdTomato-reporter mice to trace the lineage
of putative nestin-expressing cells and their progeny in the
cochleae of adult mice. Nestin, an intermediate filament found
in neural progenitor cells during early development and
adulthood, is regarded as a multipotent and neural stem cell
marker. Other investigators have reported its presence in
postnatal and young adult rodents; however, there are
discrepancies among these reports. Using lineage tracing, we
documented a robust population of tdTomato-expressing cells
and evaluated these cells at a series of adult time points.
Upon activation of the nestin promoter, tdTomato was observed
just below and medial to the inner hair cell layer. All cells
colocalized with the stem cell and cochlear-supporting-cell
marker Sox2 as well as the supporting cell and Schwann cell
marker Sox10; however, they did not colocalize with the
Schwann cell marker Krox20, spiral ganglion marker NF200, nor
glial fibrillary acidic acid (GFAP)-expressing supporting cell
marker. The cellular identity of this unique population of
tdTomato-expressing cells in the adult cochlea of
nestin-CreER(T2)/tdTomato mice remains unclear; however, these
cells may represent a type of supporting cell on the neural
aspect of the inner hair cell layer.
BibTeX:
@article{Chow2015-uv,
  author = {Chow, Cynthia L and Guo, Weixiang and Trivedi, Parul and Zhao,
Xinyu and Gubbels, Samuel P}, title = {Characterization of a unique cell population marked by
transgene expression in the adult cochlea of
nestin-CreER(T2)/tdTomato-reporter mice}, journal = {J. Comp. Neurol.}, year = {2015}, volume = {523}, number = {10}, pages = {1474--1487}, url = {http://dx.doi.org/10.1002/cne.23747}, doi = {http://dx.doi.org/10.1002/cne.23747} }
Christensen, R., Bokinsky, A., Santella, A., Wu, Y., Marquina, J., Kovacevic, I., Kumar, A., Winter, P., McCreedy, E., Mohler, W., Bao, Z., Colón-Ramos, D. and Shroff, H. An imaging and analysis toolset for the study of
Caenorhabditiselegans neurodevelopment
2015 SPIE BiOS, pp. 93340C-93340C-9  inproceedings DOI URL 
BibTeX:
@inproceedings{Christensen2015-kh,
  author = {Christensen, Ryan and Bokinsky, Alexandra and Santella, Anthony
and Wu, Yicong and Marquina, Javier and Kovacevic, Ismar and
Kumar, Abhishek and Winter, Peter and McCreedy, Evan and Mohler,
William and Bao, Zhirong and Colón-Ramos, Daniel and Shroff,
Hari}, title = {An imaging and analysis toolset for the study of
Caenorhabditiselegans neurodevelopment}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {93340C--93340C--9}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2203125}, doi = {http://dx.doi.org/10.1117/12.2082394} }
Clausen, S. and Astrup, P. Oblique laser-sheet visualization 1995 Appl. Opt.
Vol. 34(19), pp. 3800-3805 
article DOI URL 
Abstract: A laser-sheet visualization technique is demonstrated in which
the laser and camera systems are integrated into a single unit,
reducing the need for optical access to a single optical port.
The technique is based on the photographing of a plane oblique to
the camera optical axis and has been successfully applied to the
quarl region of a power-station pulverized coal burner. The
geometry of oblique photographing is presented.
BibTeX:
@article{Clausen1995-iv,
  author = {Clausen, S and Astrup, P},
  title = {Oblique laser-sheet visualization},
  journal = {Appl. Opt.},
  year = {1995},
  volume = {34},
  number = {19},
  pages = {3800--3805},
  url = {http://dx.doi.org/10.1364/AO.34.003800},
  doi = {http://dx.doi.org/10.1364/AO.34.003800}
}
Cogswell, C.J., Larkin, K.G. and Klemm, H.U. Fluorescence microtomography: multiangle image acquisition and
3D digital reconstruction
1996
Vol. 2655Electronic Imaging: Science & Technology, pp. 109-115 
inproceedings DOI URL 
Abstract: abstract We have developed a prototype fluorescence microscope
which, using tomographic image acquisition and reconstruction
techniques, can automatically combine conventional and/or
confocal image stacks taken at a number of orientations into a
single, very-high- ...
BibTeX:
@inproceedings{Cogswell1996-gg,
  author = {Cogswell, Carol J and Larkin, Kieran G and Klemm, Hanno U},
  title = {Fluorescence microtomography: multiangle image acquisition and
3D digital reconstruction}, booktitle = {Electronic Imaging: Science & Technology}, publisher = {International Society for Optics and Photonics}, year = {1996}, volume = {2655}, pages = {109--115}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1014166}, doi = {http://dx.doi.org/10.1117/12.237467} }
Conte, F., Germani, A. and Iannello, G. A Kalman filter approach for denoising and deblurring 3-D
images by multi-view data
2013 Decision and Control (CDC), 2013 IEEE 52nd Annual Conference
on, pp. 7672-7677 
inproceedings DOI URL 
Abstract: This paper introduces a novel multi-view deconvolution technique
for 3-D images. An optimal Kalman-based minimum variance
restoration algorithm is allowed to combine a series of image
samples acquired from different viewing directions. The extended
algorithm is based on the definition of a stochastic state-space
representation of the image, which embeds the description of
blurring effects and noise disturbances. The consistency of this
model gives guarantee for high restoration performances. The
extension to the data fusion is obtained by suitably including
the multi-view acquisition procedure within the representation.
The final algorithm results to be effective for improving the
resolution and the isotropy of the estimated image, as shown by
the reported numerical results.
BibTeX:
@inproceedings{Conte2013-vp,
  author = {Conte, F and Germani, A and Iannello, G},
  title = {A Kalman filter approach for denoising and deblurring 3-D
images by multi-view data}, booktitle = {Decision and Control (CDC), 2013 IEEE 52nd Annual Conference
on}, publisher = {ieeexplore.ieee.org}, year = {2013}, pages = {7672--7677}, url = {http://dx.doi.org/10.1109/CDC.2013.6761107}, doi = {http://dx.doi.org/10.1109/CDC.2013.6761107} }
Corbett, A.D. and Bub, G. Biophotonics: Bringing the living brain into focus 2015 Nat. Photonics
Vol. 9(2), pp. 80-82 
article DOI URL 
Abstract: A fast scanning light-sheet microscope that produces
multicolour, dynamic images of living tissue could provide fresh
insights into the brain's neural circuits.
BibTeX:
@article{Corbett2015-ue,
  author = {Corbett, Alexander D and Bub, Gil},
  title = {Biophotonics: Bringing the living brain into focus},
  journal = {Nat. Photonics},
  publisher = {Nature Publishing Group},
  year = {2015},
  volume = {9},
  number = {2},
  pages = {80--82},
  url = {http://dx.doi.org/10.1038/nphoton.2015.3},
  doi = {http://dx.doi.org/10.1038/nphoton.2015.3}
}
Costa, A., Candeo, A., Fieramonti, L., Valentini, G. and Bassi, A. Calcium dynamics in root cells of Arabidopsis thaliana
visualized with selective plane illumination microscopy
2013 PLoS One
Vol. 8(10), pp. e75646 
article DOI URL 
Abstract: Selective Plane Illumination Microscopy (SPIM) is an imaging
technique particularly suited for long term in-vivo analysis
of transparent specimens, able to visualize small organs or
entire organisms, at cellular and eventually even subcellular
resolution. Here we report the application of SPIM in Calcium
imaging based on Förster Resonance Energy Transfer
(FRET). Transgenic Arabidopsis plants expressing the
genetically encoded-FRET-based Ca(2+) probe Cameleon, in the
cytosol or nucleus, were used to demonstrate that SPIM enables
ratiometric fluorescence imaging at high spatial and temporal
resolution, both at tissue and single cell level. The
SPIM-FRET technique enabled us to follow nuclear and cytosolic
Ca(2+) dynamics in Arabidopsis root tip cells, deep inside the
organ, in response to different stimuli. A relevant
physiological phenomenon, namely Ca(2+) signal percolation,
predicted in previous studies, has been directly visualized.
BibTeX:
@article{Costa2013-lp,
  author = {Costa, Alex and Candeo, Alessia and Fieramonti, Luca and
Valentini, Gianluca and Bassi, Andrea}, title = {Calcium dynamics in root cells of Arabidopsis thaliana
visualized with selective plane illumination microscopy}, journal = {PLoS One}, year = {2013}, volume = {8}, number = {10}, pages = {e75646}, url = {http://dx.doi.org/10.1371/journal.pone.0075646}, doi = {http://dx.doi.org/10.1371/journal.pone.0075646} }
Costello, J.H., Colin, S.P., Gemmell, B.J., Dabiri, J.O. and Sutherland, K.R. Multi-jet propulsion organized by clonal development in a
colonial siphonophore
2015 Nat. Commun.
Vol. 6, pp. 8158 
article DOI URL 
Abstract: Physonect siphonophores are colonial cnidarians that are
pervasive predators in many neritic and oceanic ecosystems.
Physonects employ multiple, clonal medusan individuals, termed
nectophores, to propel an aggregate colony. Here we show that
developmental differences between clonal nectophores of the
physonect Nanomia bijuga produce a division of labour in
thrust and torque production that controls direction and
magnitude of whole-colony swimming. Although smaller and less
powerful, the position of young nectophores near the apex of
the nectosome allows them to dominate torque production for
turning, whereas older, larger and more powerful individuals
near the base of the nectosome contribute predominantly to
forward thrust production. The patterns we describe offer
insight into the biomechanical success of an ecologically
important and widespread colonial animal group, but, more
broadly, provide basic physical understanding of a natural
solution to multi-engine organization that may contribute to
the expanding field of underwater-distributed propulsion
vehicle design.
BibTeX:
@article{Costello2015-ry,
  author = {Costello, John H and Colin, Sean P and Gemmell, Brad J and
Dabiri, John O and Sutherland, Kelly R}, title = {Multi-jet propulsion organized by clonal development in a
colonial siphonophore}, journal = {Nat. Commun.}, publisher = {Nature Publishing Group}, year = {2015}, volume = {6}, pages = {8158}, url = {http://dx.doi.org/10.1038/ncomms9158}, doi = {http://dx.doi.org/10.1038/ncomms9158} }
Cutrale, F. and Gratton, E. Inclined selective plane illumination microscopy adaptor for
conventional microscopes
2012 Microsc. Res. Tech.
Vol. 75(11), pp. 1461-1466 
article DOI URL 
Abstract: Driven by the biological sciences, there is an increased need
for imaging modalities capable of live cell imaging with high
spatial and temporal resolution. To achieve this goal in a
comprehensive manner, three-dimensional acquisitions are
necessary. Ideal features of a modern microscope system should
include high imaging speed, high contrast ratio, low
photo-bleaching and photo-toxicity, good resolution in a 3D
context, and mosaic acquisition for large samples. Given the
importance of collecting data in live sample further increases
the technical challenges required to solve these issues. This
work presents a practical version of a microscopy method,
Selective Plane Illumination Microscopy re-introduced by
Huisken et al. (Science2004,305,1007-1009). This method is
gaining importance in the biomedical field, but its use is
limited by difficulties associated with unconventional
microscope design which employs two objectives and a
particular kind of sample preparation needed to insert the
sample between the objectives. Based on the selective plane
illumination principle but with a design similar to the Total
Internal Reflection Fluorescence microscope, Dunsby (Dunsby,
Opt Express 2008,16,20306-20316) demonstrated the oblique
plane microscope (OPM) using a single objective which uses
conventional sample preparation protocols. However, the Dunsby
instrument was not intended to be part of a commercial
microscope. In this work, we describe a system with the
advantages of OPM and that can be used as an adaptor to
commonly used microscopes, such as IX-71 Olympus, simplifying
the construction of the OPM and increasing performance of a
conventional microscope. We named our design inclined
selective plane illumination microscope (iSPIM).
BibTeX:
@article{Cutrale2012-do,
  author = {Cutrale, Francesco and Gratton, Enrico},
  title = {Inclined selective plane illumination microscopy adaptor for
conventional microscopes}, journal = {Microsc. Res. Tech.}, year = {2012}, volume = {75}, number = {11}, pages = {1461--1466}, url = {http://dx.doi.org/10.1002/jemt.22089}, doi = {http://dx.doi.org/10.1002/jemt.22089} }
De Souza, N. Method of the Year 2014 2015 Nat. Methods
Vol. 12(1), pp. 1 
article DOI URL 
Abstract: Light-sheet fluorescence microscopy can image living samples in
three dimensions with relatively low phototoxicity and at high
speed.
BibTeX:
@article{De_Souza2015-cr,
  author = {De Souza, Natalie},
  title = {Method of the Year 2014},
  journal = {Nat. Methods},
  publisher = {Nature Publishing Group},
  year = {2015},
  volume = {12},
  number = {1},
  pages = {1},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/25699311},
  doi = {http://dx.doi.org/10.1038/nmeth.3251}
}
De Vos, W.H., Beghuin, D., Schwarz, C.J., Jones, D.B., van Loon, J.J.W.A., Bereiter-Hahn, J. and Stelzer, E.H.K. Invited review article: Advanced light microscopy for
biological space research
2014 Rev. Sci. Instrum.
Vol. 85(10), pp. 101101 
article DOI URL 
Abstract: As commercial space flights have become feasible and long-term
extraterrestrial missions are planned, it is imperative that
the impact of space travel and the space environment on human
physiology be thoroughly characterized. Scrutinizing the
effects of potentially detrimental factors such as ionizing
radiation and microgravity at the cellular and tissue level
demands adequate visualization technology. Advanced light
microscopy (ALM) is the leading tool for non-destructive
structural and functional investigation of static as well as
dynamic biological systems. In recent years, technological
developments and advances in photochemistry and genetic
engineering have boosted all aspects of resolution, readout
and throughput, rendering ALM ideally suited for biological
space research. While various microscopy-based studies have
addressed cellular response to space-related environmental
stressors, biological endpoints have typically been determined
only after the mission, leaving an experimental gap that is
prone to bias results. An on-board, real-time microscopical
monitoring device can bridge this gap. Breadboards and even
fully operational microscope setups have been conceived, but
they need to be rendered more compact and versatile. Most
importantly, they must allow addressing the impact of gravity,
or the lack thereof, on physiologically relevant biological
systems in space and in ground-based simulations. In order to
delineate the essential functionalities for such a system, we
have reviewed the pending questions in space science, the
relevant biological model systems, and the state-of-the art in
ALM. Based on a rigorous trade-off, in which we recognize the
relevance of multi-cellular systems and the cellular
microenvironment, we propose a compact, but flexible concept
for space-related cell biological research that is based on
light sheet microscopy.
BibTeX:
@article{De_Vos2014-xe,
  author = {De Vos, Winnok H and Beghuin, Didier and Schwarz, Christian J
and Jones, David B and van Loon, Jack J W A and Bereiter-Hahn,
Juergen and Stelzer, Ernst H K}, title = {Invited review article: Advanced light microscopy for
biological space research}, journal = {Rev. Sci. Instrum.}, year = {2014}, volume = {85}, number = {10}, pages = {101101}, url = {http://dx.doi.org/10.1063/1.4898123}, doi = {http://dx.doi.org/10.1063/1.4898123} }
Dean, K.M. and Fiolka, R. Uniform and scalable light-sheets generated by extended focusing 2014 Opt. Express
Vol. 22(21), pp. 26141-26152 
article DOI URL 
Abstract: Light-sheet fluorescence microscopy (LSFM) affords highly
parallelized 3D imaging with optical sectioning capability and
minimal light exposure. However, using Gaussian beams for
light-sheet generation results in a trade-off between beam waist
thickness and the area over which the beam can approximate a
light-sheet. Here, we present a novel form of LSFM that uses
incoherent extended focusing to produce divergence free
light-sheets with near diffraction-limited resolution and
uniform intensity distribution along the propagation direction.
We demonstrate the imaging performance of the new technique by
volumetric imaging of beads, collagen fibers, and melanoma
cancer cells with sub-cellular resolution.
BibTeX:
@article{Dean2014-pb,
  author = {Dean, Kevin M and Fiolka, Reto},
  title = {Uniform and scalable light-sheets generated by extended focusing},
  journal = {Opt. Express},
  publisher = {Optical Society of America},
  year = {2014},
  volume = {22},
  number = {21},
  pages = {26141--26152},
  url = {http://dx.doi.org/10.1364/OE.22.026141},
  doi = {http://dx.doi.org/10.1364/OE.22.026141}
}
Dean, K.M., Roudot, P., Welf, E.S., Danuser, G. and Fiolka, R. Deconvolution-free Subcellular Imaging with Axially Swept
Light Sheet Microscopy
2015 Biophys. J.
Vol. 108(12), pp. 2807-2815 
article DOI URL 
Abstract: The use of propagation invariant Bessel beams has enabled
high-resolution subcellular light sheet fluorescence
microscopy. However, the energy within the concentric side
lobe structure of Bessel beams increases significantly with
propagation length, generating unwanted out-of-focus
fluorescence that enforces practical limits on the imaging
field of view size. Here, we present a light sheet
fluorescence microscope that achieves 390 nm isotropic
resolution and high optical sectioning strength (i.e.,
out-of-focus blur is strongly suppressed) over large field of
views, without the need for structured illumination or
deconvolution-based postprocessing. We demonstrate
simultaneous dual-color, high-contrast, and high-dynamic-range
time-lapse imaging of migrating cells in complex
three-dimensional microenvironments, three-dimensional
tracking of clathrin-coated pits, and long-term imaging
spanning >10 h and encompassing >2600 time points.
BibTeX:
@article{Dean2015-ce,
  author = {Dean, Kevin M and Roudot, Philippe and Welf, Erik S and
Danuser, Gaudenz and Fiolka, Reto}, title = {Deconvolution-free Subcellular Imaging with Axially Swept
Light Sheet Microscopy}, journal = {Biophys. J.}, year = {2015}, volume = {108}, number = {12}, pages = {2807--2815}, url = {http://dx.doi.org/10.1016/j.bpj.2015.05.013}, doi = {http://dx.doi.org/10.1016/j.bpj.2015.05.013} }
Deschout, H., Raemdonck, K., Stremersch, S., Maoddi, P., Mernier, G., Renaud, P., Jiguet, S., Hendrix, A., Bracke, M., Van den Broecke, R., Röding, M., Rudemo, M., Demeester, J., De Smedt, S.C., Strubbe, F., Neyts, K. and Braeckmans, K. On-chip light sheet illumination enables diagnostic size and
concentration measurements of membrane vesicles in biofluids
2013 Nanoscale
Vol. 6(3), pp. 1741-1747 
article DOI URL 
Abstract: Cell-derived membrane vesicles that are released in biofluids,
like blood or saliva, are emerging as potential non-invasive
biomarkers for diseases, such as cancer. Techniques capable of
measuring the size and concentration of membrane vesicles
directly in biofluids are urgently needed. Fluorescence single
particle tracking microscopy has the potential of doing exactly
that by labelling the membrane vesicles with a fluorescent label
and analysing their Brownian motion in the biofluid. However, an
unbound dye in the biofluid can cause high background intensity
that strongly biases the fluorescence single particle tracking
size and concentration measurements. While such background
intensity can be avoided with light sheet illumination, current
set-ups require specialty sample holders that are not compatible
with high-throughput diagnostics. Here, a microfluidic chip with
integrated light sheet illumination is reported, and accurate
fluorescence single particle tracking size and concentration
measurements of membrane vesicles in cell culture medium and in
interstitial fluid collected from primary human breast tumours
are demonstrated.
BibTeX:
@article{Deschout2013-sh,
  author = {Deschout, Hendrik and Raemdonck, Koen and Stremersch, Stephan
and Maoddi, Pietro and Mernier, Guillaume and Renaud, Philippe
and Jiguet, Sébastien and Hendrix, An and Bracke, Marc and
Van den Broecke, Rudy and Röding, Magnus and Rudemo, Mats
and Demeester, Jo and De Smedt, Stefaan C and Strubbe, Filip and
Neyts, Kristiaan and Braeckmans, Kevin}, title = {On-chip light sheet illumination enables diagnostic size and
concentration measurements of membrane vesicles in biofluids}, journal = {Nanoscale}, publisher = {The Royal Society of Chemistry}, year = {2013}, volume = {6}, number = {3}, pages = {1741--1747}, url = {http://pubs.rsc.org/en/Content/ArticleLanding/2014/NR/C3NR04432G}, doi = {http://dx.doi.org/10.1039/C3NR04432G} }
Desmaison, A., Lorenzo, C., Rouquette, J., Ducommun, B. and Lobjois, V. A versatile sample holder for single plane illumination
microscopy
2013 J. Microsc.
Vol. 251(2), pp. 128-132 
article DOI URL 
Abstract: Single Plane Illumination Microscopy is an emerging and
powerful technology for live imaging of whole living
organisms. However, sample handling that relies on specimen
embedding in agarose or gel is often a key limitation,
especially for time-lapse monitoring. To address this issue,
we developed a new concept for a holder device allowing us to
prepare a sample container made of hydrogel. The production
process of this holder is based on 3D printing of both a frame
and casting devices. The simplicity of production and the
advantages of this versatile new sample holder are shown with
time-lapse recording of multicellular tumour spheroid growth.
More importantly, we also show that cell division is not
impaired in contrast to what is observed with gel embedding.
The benefit of this new holder for other sample types,
applications and experiments remains to be evaluated, but this
innovative concept of fully customizable sample holder
preparation potentially represents a major step forward to
facilitate the large diffusion of single plane illumination
microscopy technology.
BibTeX:
@article{Desmaison2013-vp,
  author = {Desmaison, Annack and Lorenzo, Corinne and Rouquette,
Jacques and Ducommun, Bernard and Lobjois, Valérie}, title = {A versatile sample holder for single plane illumination
microscopy}, journal = {J. Microsc.}, year = {2013}, volume = {251}, number = {2}, pages = {128--132}, url = {http://dx.doi.org/10.1111/jmi.12051}, doi = {http://dx.doi.org/10.1111/jmi.12051} }
Dobosz, M., Ntziachristos, V., Scheuer, W. and Strobel, S. Multispectral fluorescence ultramicroscopy: three-dimensional
visualization and automatic quantification of tumor
morphology, drug penetration, and antiangiogenic treatment
response
2014 Neoplasia
Vol. 16(1), pp. 1-13 
article DOI URL 
Abstract: Classic histology still represents the gold standard in tumor
tissue analytics. However, two-dimensional analysis of single
tissue slides does not provide a representative overview of
the inhomogeneous tumor physiology, and a detailed analysis of
complex three-dimensional structures is not feasible with this
technique. To overcome this problem, we applied multispectral
fluorescence ultramicroscopy (UM) to the field of tumor
analysis. Optical sectioning of cleared tumor specimen
provides the possibility to three-dimensionally acquire
relevant tumor parameters on a cellular resolution. To analyze
the virtual UM tumor data sets, we created a novel set of
algorithms enabling the fully automatic segmentation and
quantification of multiple tumor parameters. This new
postmortem imaging technique was applied to determine the
therapeutic treatment effect of bevacizumab on the vessel
architecture of orthotopic KPL-4 breast cancer xenografts at
different time points. A significant reduction of the vessel
volume, number of vessel segments, and branching points in the
tumor periphery was already detectable 1 day after initiation
of treatment. These parameters remained virtually unchanged in
the center of the tumor. Furthermore, bevacizumab-induced
vessel normalization and reduction in vascular permeability
diminished the penetration behavior of trastuzumab-Alexa 750
into tumor tissue. Our results demonstrated that this
newimaging method enables the three-dimensional visualization
and fully automatic quantification of multiple tumor
parameters and drug penetration on a cellular level.
Therefore,UM is a valuable tool for cancer research and drug
development. It bridges the gap between common macroscopic and
microscopic imaging modalities and opens up new
three-dimensional (3D) insights in tumor biology.
BibTeX:
@article{Dobosz2014-jb,
  author = {Dobosz, Michael and Ntziachristos, Vasilis and Scheuer, Werner
and Strobel, Steffen}, title = {Multispectral fluorescence ultramicroscopy: three-dimensional
visualization and automatic quantification of tumor
morphology, drug penetration, and antiangiogenic treatment
response}, journal = {Neoplasia}, year = {2014}, volume = {16}, number = {1}, pages = {1--13}, url = {http://www.ncbi.nlm.nih.gov/pubmed/24563615}, doi = {http://dx.doi.org/10.1593/neo.131848} }
Dodt, H.-U., Leischner, U., Schierloh, A., Jährling, N., Mauch, C.P., Deininger, K., Deussing, J.M., Eder, M., Zieglgänsberger, W. and Becker, K. Ultramicroscopy: three-dimensional visualization of neuronal
networks in the whole mouse brain
2007 Nat. Methods
Vol. 4(4), pp. 331-336 
article DOI URL 
Abstract: Visualizing entire neuronal networks for analysis in the
intact brain has been impossible up to now. Techniques like
computer tomography or magnetic resonance imaging (MRI) do not
yield cellular resolution, and mechanical slicing procedures
are insufficient to achieve high-resolution reconstructions in
three dimensions. Here we present an approach that allows
imaging of whole fixed mouse brains. We modified
'ultramicroscopy' by combining it with a special procedure to
clear tissue. We show that this new technique allows optical
sectioning of fixed mouse brains with cellular resolution and
can be used to detect single GFP-labeled neurons in excised
mouse hippocampi. We obtained three-dimensional (3D) images of
dendritic trees and spines of populations of CA1 neurons in
isolated hippocampi. Also in fruit flies and in mouse embryos,
we were able to visualize details of the anatomy by imaging
autofluorescence. Our method is ideally suited for
high-throughput phenotype screening of transgenic mice and
thus will benefit the investigation of disease models.
BibTeX:
@article{Dodt2007-sf,
  author = {Dodt, Hans-Ulrich and Leischner, Ulrich and Schierloh, Anja
and Jährling, Nina and Mauch, Christoph Peter and
Deininger, Katrin and Deussing, Jan Michael and Eder, Matthias
and Zieglgänsberger, Walter and Becker, Klaus}, title = {Ultramicroscopy: three-dimensional visualization of neuronal
networks in the whole mouse brain}, journal = {Nat. Methods}, year = {2007}, volume = {4}, number = {4}, pages = {331--336}, url = {http://dx.doi.org/10.1038/nmeth1036}, doi = {http://dx.doi.org/10.1038/nmeth1036} }
Dong, D., Arranz, A., Zhu, S., Yang, Y., Shi, L., Wang, J., Shen, C., Tian, J. and Ripoll, J. Vertically scanned laser sheet microscopy 2014 J. Biomed. Opt.
Vol. 19(10), pp. 106001 
article DOI URL 
Abstract: Laser sheet microscopy is a widely used imaging technique for
imaging the three-dimensional distribution of a fluorescence
signal in fixed tissue or small organisms. In laser sheet
microscopy, the stripe artifacts caused by high absorption or
high scattering structures are very common, greatly affecting
image quality. To solve this problem, we report here a
two-step procedure which consists of continuously acquiring
laser sheet images while vertically displacing the sample, and
then using the variational stationary noise remover (VSNR)
method to further reduce the remaining stripes. Images from a
cleared murine colon acquired with a vertical scan are
compared with common stitching procedures demonstrating that
vertically scanned light sheet microscopy greatly improves the
performance of current light sheet microscopy approaches
without the need for complex changes to the imaging setup and
allows imaging of elongated samples, extending the field of
view in the vertical direction.
BibTeX:
@article{Dong2014-nv,
  author = {Dong, Di and Arranz, Alicia and Zhu, Shouping and Yang, Yujie
and Shi, Liangliang and Wang, Jun and Shen, Chen and Tian, Jie
and Ripoll, Jorge}, title = {Vertically scanned laser sheet microscopy}, journal = {J. Biomed. Opt.}, year = {2014}, volume = {19}, number = {10}, pages = {106001}, url = {http://dx.doi.org/10.1117/1.JBO.19.10.106001}, doi = {http://dx.doi.org/10.1117/1.JBO.19.10.106001} }
Dunsby, C. Optically sectioned imaging by oblique plane microscopy 2008 Opt. Express
Vol. 16(25), pp. 20306-20316 
article URL 
Abstract: This paper describes a new optically sectioning microscopy
technique based on oblique selective plane illumination
combined with oblique imaging. This method differs from
previous selective plane illumination techniques as the same
high numerical aperture lens is used to both illuminate and
image the specimen. Initial results obtained using fluorescent
pollen grains are presented, together with a measurement of
the resolution of the system and an analysis of the potential
performance of future systems. Since only the plane of the
specimen that is being imaged is illuminated, this technique
is particularly suited to time-lapse 3-D imaging of sensitive
biological systems where photobleaching and phototoxicity must
be kept to a minimum, and it could also be applied to image
microfluidic technology for lab-on-a-chip, cytometry and other
applications.
BibTeX:
@article{Dunsby2008-kh,
  author = {Dunsby, C},
  title = {Optically sectioned imaging by oblique plane microscopy},
  journal = {Opt. Express},
  year = {2008},
  volume = {16},
  number = {25},
  pages = {20306--20316},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/19065169}
}
Ebeling, C.G. and Jorgensen, E.M. Two views on light sheets 2013 Nat. Biotechnol.
Vol. 31(11), pp. 992-993 
article DOI URL 
BibTeX:
@article{Ebeling2013-re,
  author = {Ebeling, Carl G and Jorgensen, Erik M},
  title = {Two views on light sheets},
  journal = {Nat. Biotechnol.},
  year = {2013},
  volume = {31},
  number = {11},
  pages = {992--993},
  url = {http://dx.doi.org/10.1038/nbt.2739},
  doi = {http://dx.doi.org/10.1038/nbt.2739}
}
Eisenstein, M. Pump up the volume 2014 Nat. Methods
Vol. 12(1), pp. 19-22 
article DOI URL 
Abstract: Light-sheet fluorescence microscopy techniques are enabling
researchers to achieve dynamic, long-term imaging and
three-dimensional reconstruction of specimens ranging from
single cells to whole embryos.
BibTeX:
@article{Eisenstein2014-qn,
  author = {Eisenstein, Michael},
  title = {Pump up the volume},
  journal = {Nat. Methods},
  publisher = {Nature Publishing Group},
  year = {2014},
  volume = {12},
  number = {1},
  pages = {19--22},
  url = {http://dx.doi.org/10.1038/nmeth.3220},
  doi = {http://dx.doi.org/10.1038/nmeth.3220}
}
Engelbrecht, C.J., Greger, K., Reynaud, E.G., Krzic, U., Colombelli, J. and Stelzer, E.H. Three-dimensional laser microsurgery in light-sheet based
microscopy (SPIM)
2007 Opt. Express
Vol. 15(10), pp. 6420-6430 
article URL 
Abstract: Advances in the life sciences rely on the ability to observe
dynamic processes in live systems and in environments that
mimic in-vivo situations. Therefore, new methodological
developments have to provide environments that resemble
physiologically and clinically relevant conditions as closely
as possible. In this work, plasma-induced laser nanosurgery
for three-dimensional sample manipulation and sample
perturbation is combined with optically sectioning light-sheet
based fluorescence microscopy (SPIM) and applied to
three-dimensional biological model systems. This means: a)
working with a biological system that is not confined to
essentially two dimensions like cell cultures on cover
glasses, b) gaining intrinsic optical sectioning capabilities
by an efficient three-dimensional fluorescence imaging system,
and c) using arbitrarily-shaped three-dimensional
ablation-patterns by a plasma-induced laser ablation system
that prevent damage to surrounding tissues. Spatial levels in
our biological applications range from sub-microns during
delicate ablation of single microtubules over the confined
disruption of cell membranes in an MDCK-cyst to the
macroscopic cutting of a millimeter-sized Zebrafish caudal fin
with arbitrary three-dimensional ablation patterns. Dynamic
processes like laser-induced hemocyte migration can be studied
with our SPIM-microscalpel in intact, live embryos.
BibTeX:
@article{Engelbrecht2007-gf,
  author = {Engelbrecht, Christoph J and Greger, Klaus and Reynaud,
Emmanuel G and Krzic, Uros and Colombelli, Julien and Stelzer,
Ernst H}, title = {Three-dimensional laser microsurgery in light-sheet based
microscopy (SPIM)}, journal = {Opt. Express}, year = {2007}, volume = {15}, number = {10}, pages = {6420--6430}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19546948} }
Engelbrecht, C.J., Johnston, R.S., Seibel, E.J. and Helmchen, F. Ultra-compact fiber-optic two-photon microscope for functional
fluorescence imaging in vivo
2008 Opt. Express
Vol. 16(8), pp. 5556-5564 
article URL 
Abstract: We present a small, lightweight two-photon fiberscope and
demonstrate its suitability for functional imaging in the
intact brain. Our device consists of a hollow-core photonic
crystal fiber for efficient delivery of near-IR femtosecond
laser pulses, a spiral fiber-scanner for resonant beam
steering, and a gradient-index lens system for fluorescence
excitation, dichroic beam splitting, and signal collection.
Fluorescence light is remotely detected using a standard
photomultiplier tube. All optical components have 1 mm
dimensions and the microscope's headpiece weighs only 0.6
grams. The instrument achieves micrometer resolution at frame
rates of typically 25 Hz with a field-of-view of up to 200
microns. We demonstrate functional imaging of calcium signals
in Purkinje cell dendrites in the cerebellum of anesthetized
rats. The microscope will be easily portable by a rat or mouse
and thus should enable functional imaging in freely behaving
animals.
BibTeX:
@article{Engelbrecht2008-mn,
  author = {Engelbrecht, Christoph J and Johnston, Richard S and Seibel,
Eric J and Helmchen, Fritjof}, title = {Ultra-compact fiber-optic two-photon microscope for functional
fluorescence imaging in vivo}, journal = {Opt. Express}, year = {2008}, volume = {16}, number = {8}, pages = {5556--5564}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18542658} }
Engelbrecht, C.J. and Stelzer, E.H. Resolution enhancement in a light-sheet-based microscope
(SPIM)
2006 Opt. Lett.
Vol. 31(10), pp. 1477-1479 
article URL 
Abstract: Light-sheet-based microscopy [single-plane illumination
microscope (SPIM)] performs very well at low numerical
apertures. It complements conventional (FM), confocal (CFM),
and two-photon fluorescence microscopy (2hnu-FM) currently
used in modern life sciences. Lateral and axial SPIM point
spread function (PSF) extents are measured by using
fluorescent beads to determine the 3D resolution. The results
are compared with values derived from an analytical theory and
numerical simulations. The discrepancies are found to be less
than 5 The axial extent of a SPIM-PSF (10x/0.3 W) is
approximately 5.7 microm. This value is almost a factor of 2
smaller than in CFM, more than 2.5 times smaller than in FM,
and more than three times smaller than in 2hnu-FM. SPIM
outperforms 2hnu-FM and FM, while CFM has a better axial
resolution at NAs above 0.8.
BibTeX:
@article{Engelbrecht2006-vm,
  author = {Engelbrecht, Christoph J and Stelzer, Ernst H},
  title = {Resolution enhancement in a light-sheet-based microscope
(SPIM)}, journal = {Opt. Lett.}, year = {2006}, volume = {31}, number = {10}, pages = {1477--1479}, url = {http://www.ncbi.nlm.nih.gov/pubmed/16642144} }
Engelbrecht, C.J., Voigt, F. and Helmchen, F. Miniaturized selective plane illumination microscopy for
high-contrast in vivo fluorescence imaging
2010 Opt. Lett.
Vol. 35(9), pp. 1413-1415 
article DOI URL 
Abstract: Light-sheet-based fluorescence imaging techniques rely on
simultaneous excitation of a single optical plane and thus
permit high-contrast optically sectioned imaging of extended
tissue samples. Here, we introduce a miniaturized fiber-optic
implementation of a selective plane-illumination microscope
(miniSPIM). The excitation light was delivered through a
single-mode optical fiber, and a light-sheet was created with
a cylindrical gradient-index lens and a right-angle
microprism. Fluorescence emission was collected orthogonally
to the light-sheet through a gradient-index lens assembly and
a coherent fiber bundle. The end face of the fiber bundle was
imaged onto a charge-coupled device camera. The spatial
resolutions of the miniSPIM were 3.2 microm laterally and 5.1
microm axially. Images of fluorescent beads and neurons in
mouse neocortex exhibited superior axial resolution and
contrast in the miniSPIM-mode compared to images recorded in
epi-illumination mode. The miniSPIM may thus enable novel in
vivo imaging approaches.
BibTeX:
@article{Engelbrecht2010-il,
  author = {Engelbrecht, Christoph J and Voigt, Fabian and Helmchen,
Fritjof}, title = {Miniaturized selective plane illumination microscopy for
high-contrast in vivo fluorescence imaging}, journal = {Opt. Lett.}, year = {2010}, volume = {35}, number = {9}, pages = {1413--1415}, url = {http://dx.doi.org/10.1364/OL.35.001413}, doi = {http://dx.doi.org/10.1364/OL.35.001413} }
Ermolayev, V., Friedrich, M., Nozadze, R., Cathomen, T., Klein, M.A., Harms, G.S. and Flechsig, E. Ultramicroscopy reveals axonal transport impairments in
cortical motor neurons at prion disease
2009 Biophys. J.
Vol. 96(8), pp. 3390-3398 
article DOI URL 
Abstract: The functional imaging of neuronal circuits of the central
nervous system is crucial for phenotype screenings or
investigations of defects in neurodegenerative disorders.
Current techniques yield either low penetration depth, yield
poor resolution, or are restricted by the age of the animals.
Here, we present a novel ultramicroscopy protocol for
fluorescence imaging and three-dimensional reconstruction in
the central nervous system of adult mice. In combination with
tracing as a functional assay for axonal transport,
retrogradely labeled descending motor neurons were visualized
with >4 mm penetration depth. The analysis of the motor cortex
shortly before the onset of clinical prion disease revealed
that >80% neurons have functional impairments in axonal
transport. Our study provides evidence that prion disease is
associated with severe axonal transport defects in the
cortical motor neurons and suggests a novel mechanism for
prion-mediated neurodegeneration.
BibTeX:
@article{Ermolayev2009-bn,
  author = {Ermolayev, Vladimir and Friedrich, Mike and Nozadze, Revaz and
Cathomen, Toni and Klein, Michael A and Harms, Gregory S and
Flechsig, Eckhard}, title = {Ultramicroscopy reveals axonal transport impairments in
cortical motor neurons at prion disease}, journal = {Biophys. J.}, year = {2009}, volume = {96}, number = {8}, pages = {3390--3398}, url = {http://dx.doi.org/10.1016/j.bpj.2009.01.032}, doi = {http://dx.doi.org/10.1016/j.bpj.2009.01.032} }
Ertürk, A., Becker, K., Jährling, N., Mauch, C.P., Hojer, C.D., Egen, J.G., Hellal, F., Bradke, F., Sheng, M. and Dodt, H.-U. Three-dimensional imaging of solvent-cleared organs using
3DISCO
2012 Nat. Protoc.
Vol. 7(11), pp. 1983-1995 
article DOI URL 
Abstract: The examination of tissue histology by light microscopy is a
fundamental tool for investigating the structure and function
of organs under normal and disease states. Many current
techniques for tissue sectioning, imaging and analysis are
time-consuming, and they present major limitations for 3D
tissue reconstruction. The introduction of methods to achieve
the optical clearing and subsequent light-sheet laser scanning
of entire transparent organs without sectioning represents a
major advance in the field. We recently developed a highly
reproducible and versatile clearing procedure called 3D
imaging of solvent-cleared organs, or 3DISCO, which is
applicable to diverse tissues including brain, spinal cord,
immune organs and tumors. Here we describe a detailed protocol
for performing 3DISCO and present its application to various
microscopy techniques, including example results from various
mouse tissues. The tissue clearing takes as little as 3 h, and
imaging can be completed in 45 min. 3DISCO is a powerful
technique that offers 3D histological views of tissues in a
fraction of the time and labor required to complete standard
histology studies.
BibTeX:
@article{Erturk2012-am,
  author = {Ertürk, Ali and Becker, Klaus and Jährling, Nina
and Mauch, Christoph P and Hojer, Caroline D and Egen, Jackson
G and Hellal, Farida and Bradke, Frank and Sheng, Morgan and
Dodt, Hans-Ulrich}, title = {Three-dimensional imaging of solvent-cleared organs using
3DISCO}, journal = {Nat. Protoc.}, year = {2012}, volume = {7}, number = {11}, pages = {1983--1995}, url = {http://dx.doi.org/10.1038/nprot.2012.119}, doi = {http://dx.doi.org/10.1038/nprot.2012.119} }
Fahrbach, F.O., Gurchenkov, V., Alessandri, K., Nassoy, P. and Rohrbach, A. Self-reconstructing sectioned Bessel beams offer submicron
optical sectioning for large fields of view in light-sheet
microscopy
2013 Opt. Express
Vol. 21(9), pp. 11425-11440 
article DOI URL 
Abstract: One of main challenges in light-sheet microscopy is to design
the light-sheet as extended and thin as possible--extended to
cover a large field of view, thin to optimize resolution and
contrast. However, a decrease of the beam's waist also
decreases the illumination beam's depth of field. Here, we
introduce a new kind of beam that we call sectioned Bessel
beam. These beams can be generated by blocking opposite
sections of the beam's angular spectrum. In combination with
confocal-line detection the optical sectioning performance of
the light-sheet can be decoupled from the depth of field of
the illumination beam. By simulations and experiments we
demonstrate that these beams exhibit self-reconstruction
capabilities and penetration depths into thick scattering
media equal to those of conventional Bessel beams. We applied
sectioned Bessel beams to illuminate tumor multicellular
spheroids and prove the increase in contrast. Sectioned Bessel
beams turn out to be highly advantageous for the investigation
of large strongly scattering samples in a light-sheet
microscope.
BibTeX:
@article{Fahrbach2013-gu,
  author = {Fahrbach, Florian O and Gurchenkov, Vasily and Alessandri,
Kevin and Nassoy, Pierre and Rohrbach, Alexander}, title = {Self-reconstructing sectioned Bessel beams offer submicron
optical sectioning for large fields of view in light-sheet
microscopy}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {9}, pages = {11425--11440}, url = {http://dx.doi.org/10.1364/OE.21.011425}, doi = {http://dx.doi.org/10.1364/OE.21.011425} }
Fahrbach, F.O., Gurchenkov, V., Alessandri, K., Nassoy, P. and Rohrbach, A. Light-sheet microscopy in thick media using scanned Bessel
beams and two-photon fluorescence excitation
2013 Opt. Express
Vol. 21(11), pp. 13824-13839 
article DOI URL 
Abstract: In this study we show that it is possible to successfully
combine the benefits of light-sheet microscopy,
self-reconstructing Bessel beams and two-photon fluorescence
excitation to improve imaging in large, scattering media such
as cancer cell clusters. We achieved a nearly two-fold
increase in axial image resolution and 5-10 fold increase in
contrast relative to linear excitation with Bessel beams. The
light-sheet penetration depth could be increased by a factor
of 3-5 relative to linear excitation with Gaussian beams.
These finding arise from both experiments and computer
simulations. In addition, we provide a theoretical description
of how these results are composed. We investigated the change
of image quality along the propagation direction of the
illumination beams both for clusters of spheres and tumor
multicellular spheroids. The results reveal that light-sheets
generated by pulsed near-infrared Bessel beams and two photon
excitation provide the best image resolution, contrast at both
a minimum amount of artifacts and signal degradation along the
propagation of the beam into the sample.
BibTeX:
@article{Fahrbach2013-vg,
  author = {Fahrbach, Florian O and Gurchenkov, Vasily and Alessandri,
Kevin and Nassoy, Pierre and Rohrbach, Alexander}, title = {Light-sheet microscopy in thick media using scanned Bessel
beams and two-photon fluorescence excitation}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {11}, pages = {13824--13839}, url = {http://dx.doi.org/10.1364/OE.21.013824}, doi = {http://dx.doi.org/10.1364/OE.21.013824} }
Fahrbach, F.O. and Rohrbach, A. Propagation stability of self-reconstructing Bessel beams
enables contrast-enhanced imaging in thick media
2012 Nat. Commun.
Vol. 3, pp. 632 
article DOI URL 
Abstract: Laser beams that can self-reconstruct their initial beam
profile even in the presence of massive phase perturbations
are able to propagate deeper into inhomogeneous media. This
ability has crucial advantages for light sheet-based
microscopy in thick media, such as cell clusters, embryos,
skin or brain tissue or plants, as well as scattering
synthetic materials. A ring system around the central
intensity maximum of a Bessel beam enables its
self-reconstruction, but at the same time illuminates
out-of-focus regions and deteriorates image contrast. Here we
present a detection method that minimizes the negative effect
of the ring system. The beam's propagation stability along one
straight line enables the use of a confocal line principle,
resulting in a significant increase in image contrast. The
axial resolution could be improved by nearly 100% relative to
the standard light-sheet techniques using scanned Gaussian
beams, while demonstrating self-reconstruction also for high
propagation depths.
BibTeX:
@article{Fahrbach2012-fb,
  author = {Fahrbach, Florian O and Rohrbach, Alexander},
  title = {Propagation stability of self-reconstructing Bessel beams
enables contrast-enhanced imaging in thick media}, journal = {Nat. Commun.}, year = {2012}, volume = {3}, pages = {632}, url = {http://dx.doi.org/10.1038/ncomms1646}, doi = {http://dx.doi.org/10.1038/ncomms1646} }
Fahrbach, F.O. and Rohrbach, A. A line scanned light-sheet microscope with phase shaped
self-reconstructing beams
2010 Opt. Express
Vol. 18(23), pp. 24229-24244 
article DOI URL 
Abstract: We recently demonstrated that Microscopy with
Self-Reconstructing Beams (MISERB) increases both image
quality and penetration depth of illumination beams in
strongly scattering media. Based on the concept of line
scanned light-sheet microscopy, we present an add-on module to
a standard inverted microscope using a scanned beam that is
shaped in phase and amplitude by a spatial light modulator. We
explain technical details of the setup as well as of the
holograms for the creation, positioning and scaling of static
light-sheets, Gaussian beams and Bessel beams. The comparison
of images from identical sample areas illuminated by different
beams allows a precise assessment of the interconnection
between beam shape and image quality. The superior propagation
ability of Bessel beams through inhomogeneous media is
demonstrated by measurements on various scattering media.
BibTeX:
@article{Fahrbach2010-no,
  author = {Fahrbach, Florian O and Rohrbach, Alexander},
  title = {A line scanned light-sheet microscope with phase shaped
self-reconstructing beams}, journal = {Opt. Express}, year = {2010}, volume = {18}, number = {23}, pages = {24229--24244}, url = {http://dx.doi.org/10.1364/OE.18.024229}, doi = {http://dx.doi.org/10.1364/OE.18.024229} }
Fahrbach, F.O., Simon, P. and Rohrbach, A. Microscopy with self-reconstructing beams 2010 Nat. Photonics
Vol. 4(11), pp. 780-785 
article DOI URL 
Abstract: A prototype microscope built with self-reconstructing Bessel
beams is shown to be able to reduce scattering artifacts as well
as increase image quality and penetration depth in
three-dimensional inhomogeneous opaque media.
BibTeX:
@article{Fahrbach2010-zj,
  author = {Fahrbach, Florian O and Simon, Philipp and Rohrbach, Alexander},
  title = {Microscopy with self-reconstructing beams},
  journal = {Nat. Photonics},
  publisher = {Nature Publishing Group},
  year = {2010},
  volume = {4},
  number = {11},
  pages = {780--785},
  url = {http://dx.doi.org/10.1038/nphoton.2010.204},
  doi = {http://dx.doi.org/10.1038/nphoton.2010.204}
}
Fahrbach, F.O., Voigt, F.F., Schmid, B., Helmchen, F. and Huisken, J. Rapid 3D light-sheet microscopy with a tunable lens 2013 Opt. Express
Vol. 21(18), pp. 21010-21026 
article DOI URL 
Abstract: The in-vivo investigation of highly dynamic biological samples,
for example the beating zebrafish heart, requires high-speed
volume imaging techniques. Light-sheet microscopy is ideal for
such samples as it records high-contrast images of entire planes
within large samples at once. However, in order to obtain images
of different planes, it has been necessary to move the sample
relative to the fixed focal plane of the detection objective
lens. This mechanical movement limits speed, precision and may be
harmful to the sample. We have built a light-sheet microscope
that uses remote focusing with an electrically tunable lens
(ETL). Without moving specimen or objective we have thereby
achieved flexible volume imaging at much higher speeds than
previously reported. Our high-speed microscope delivers 3D
snapshots of sensitive biological samples. As an example, we
imaged 17 planes within a beating zebrafish heart at 510 frames
per second, equivalent to 30 volume scans per second. Movements,
shape changes and signals across the entire volume can be
followed which has been impossible with existing reconstruction
techniques.
BibTeX:
@article{Fahrbach2013-pr,
  author = {Fahrbach, Florian O and Voigt, Fabian F and Schmid, Benjamin and
Helmchen, Fritjof and Huisken, Jan}, title = {Rapid 3D light-sheet microscopy with a tunable lens}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {18}, pages = {21010--21026}, url = {http://dx.doi.org/10.1364/OE.21.021010}, doi = {http://dx.doi.org/10.1364/OE.21.021010} }
Ferrario, L. Studio delle dinamiche del calcio nell'Arabidopsis thaliana con
microscopia SPIM
2013   mastersthesis URL 
Abstract: Negli ultimi cinquant'anni, lo studio delle dinamiche della
concentrazione intracellulare del Ca2+ é aumentato in modo
esponenziale, diventando un ambito di ricerca fondamentale della
biologia [27]. Infatti, il segnale del Ca2+ risulta piú
importante rispetto a quello di ...
BibTeX:
@mastersthesis{Ferrario2013-ky,
  author = {Ferrario, L},
  title = {Studio delle dinamiche del calcio nell'Arabidopsis thaliana con
microscopia SPIM}, publisher = {politesi.polimi.it}, year = {2013}, url = {https://www.politesi.polimi.it/handle/10589/80985} }
Fickentscher, R., Struntz, P. and Weiss, M. Mechanical cues in the early embryogenesis of Caenorhabditis
elegans
2013 Biophys. J.
Vol. 105(8), pp. 1805-1811 
article DOI URL 
Abstract: Biochemical signaling pathways in developmental processes have
been extensively studied, yet the role of mechanical cues
during embryogenesis is much less explored. Here we have used
selective plane illumination microscopy in combination with a
simple mechanical model to quantify and rationalize cell
motion during early embryogenesis of the small nematode
Caenorhabditis elegans. As a result, we find that cell
organization in the embryo until gastrulation is well
described by a purely mechanical model that predicts cells to
assume positions in which they face the least repulsive
interactions from other cells and the embryo's egg shell. Our
findings therefore suggest that mechanical interactions are
key for a rapid and robust cellular arrangement during early
embryogenesis of C. elegans.
BibTeX:
@article{Fickentscher2013-ni,
  author = {Fickentscher, Rolf and Struntz, Philipp and Weiss, Matthias},
  title = {Mechanical cues in the early embryogenesis of Caenorhabditis
elegans}, journal = {Biophys. J.}, year = {2013}, volume = {105}, number = {8}, pages = {1805--1811}, url = {http://dx.doi.org/10.1016/j.bpj.2013.09.005}, doi = {http://dx.doi.org/10.1016/j.bpj.2013.09.005} }
Friedrich, M., Gan, Q., Ermolayev, V. and Harms, G.S. STED-SPIM: Stimulated emission depletion improves sheet
illumination microscopy resolution
2011 Biophys. J.
Vol. 100(8), pp. L43-5 
article DOI URL 
Abstract: We demonstrate the first, to our knowledge, integration of
stimulated emission depletion (STED) with selective plane
illumination microscopy (SPIM). Using this method, we were
able to obtain up to 60% improvements in axial resolution
with lateral resolution enhancements in control samples and
zebrafish embryos. The integrated STED-SPIM method combines
the advantages of SPIM with the resolution enhancement of
STED, and thus provides a method for fast, high-resolution
imaging with >100 $m deep penetration into biological
tissue.
BibTeX:
@article{Friedrich2011-dj,
  author = {Friedrich, Mike and Gan, Qiang and Ermolayev, Vladimir and
Harms, Gregory S}, title = {STED-SPIM: Stimulated emission depletion improves sheet
illumination microscopy resolution}, journal = {Biophys. J.}, year = {2011}, volume = {100}, number = {8}, pages = {L43--5}, url = {http://dx.doi.org/10.1016/j.bpj.2010.12.3748}, doi = {http://dx.doi.org/10.1016/j.bpj.2010.12.3748} }
Friedrich, M., Nozadze, R., Gan, Q., Zelman-Femiak, M., Ermolayev, V., Wagner, T.U. and Harms, G.S. Detection of single quantum dots in model organisms with sheet
illumination microscopy
2009 Biochem. Biophys. Res. Commun.
Vol. 390(3), pp. 722-727 
article DOI URL 
Abstract: Single-molecule detection and tracking is important for
observing biomolecule interactions in the microenvironment.
Here we report selective plane illumination microscopy (SPIM)
with single-molecule detection in living organisms, which
enables fast imaging and single-molecule tracking and optical
penetration beyond 300 microm. We detected single nanocrystals
in Drosophila larvae and zebrafish embryo. We also report our
first tracking of single quantum dots during zebrafish
development, which displays a transition from flow to confined
motion prior to the blastula stage. The new SPIM setup
represents a new technique, which enables fast single-molecule
imaging and tracking in living systems.
BibTeX:
@article{Friedrich2009-po,
  author = {Friedrich, Mike and Nozadze, Revaz and Gan, Qiang and
Zelman-Femiak, Monika and Ermolayev, Vladimir and Wagner, Toni
U and Harms, Gregory S}, title = {Detection of single quantum dots in model organisms with sheet
illumination microscopy}, journal = {Biochem. Biophys. Res. Commun.}, year = {2009}, volume = {390}, number = {3}, pages = {722--727}, url = {http://dx.doi.org/10.1016/j.bbrc.2009.10.036}, doi = {http://dx.doi.org/10.1016/j.bbrc.2009.10.036} }
Friedrich, M., Nozadze, R., de Keijzer, S., Steinmeyer, R., Ermolayev, V. and Harms, G.S. Detection of Single Quantum Dots in Model Systems with Sheet
Illumination Microscopy
2011 J. Fluoresc.  article DOI URL 
Abstract: Single molecule detection and tracking provides at times the
only possible method to observe the interactions of low
numbers of biomolecules, inlcuding DNA, receptors and signal
mediating proteins in living systems. However, most existing
imaging methods do not enable both high sensitivity and
non-invasive imaging of large specimens. In this study we
report a new setup for selective plane illumination microscopy
(SPIM), which enables fast imaging and single molecule
tracking with the resolution of confocal microscopy and the
optical penetration beyond 300 $m. We detect and report
our instrumental figures of merit, control values of
fluorescence properties of single nano crystals in comparison
to both standard widefield configurations, and also values of
nanocrystals in multicellular ``fruiting bodies'' of
Dictyostelium, an excellent control as a model developmental
system. In the Dictyostelium , we also report some of our
first tracking of single nanocrystals with SPIM. The new SPIM
setup represents a new technique, which enables fast single
molecule imaging and tracking in living systems.
BibTeX:
@article{Friedrich2011-wr,
  author = {Friedrich, Mike and Nozadze, Revaz and de Keijzer, Sandra and
Steinmeyer, Ralf and Ermolayev, Vladimir and Harms, Gregory S}, title = {Detection of Single Quantum Dots in Model Systems with Sheet
Illumination Microscopy}, journal = {J. Fluoresc.}, year = {2011}, url = {http://dx.doi.org/10.1007/s10895-011-0966-4}, doi = {http://dx.doi.org/10.1007/s10895-011-0966-4} }
Fuchs, E., Jaffe, J., Long, R. and Azam, F. Thin laser light sheet microscope for microbial oceanography 2002 Opt. Express
Vol. 10(2), pp. 145-154 
article DOI URL 
Abstract: Despite a growing need, oceanographers are limited by existing
technological constrains and are unable to observe aquatic
microbes in their natural setting. In order to provide a
simple and easy to implement solution for such studies, a new
Thin Light Sheet Microscope (TLSM) has been developed. The
TLSM utilizes a well-defined sheet of laser light, which has a
narrow (23 micron) axial dimension over a 1 mm x 1 mm field of
view. This light sheet is positioned precisely within the
depth of field of the microscope's objective lens. The
technique thus utilizes conventional microscope optics but
replaces the illumination system. The advantages of the TLSM
are two-fold: First, it concentrates light only where
excitation is needed, thus maximizing the efficiency of the
illumination source. Secondly, the TLSM maximizes image
sharpness while at the same time minimizing the level of
background noise. Particles that are not located within the
objective's depth of field are not illuminated and therefore
do not contribute to an out-of-focus image. Images from a
prototype system that used SYBR Green I fluorescence stain in
order to localize single bacteria are reported. The bacteria
were in a relatively large and undisturbed volume of 4ml,
which contained natural seawater. The TLSM can be used for
fresh water studies of bacteria with no modification. The
microscope permits the observation of interactions at the
microscale and has potential to yield insights into how
microbes structure pelagic ecosystems.
BibTeX:
@article{Fuchs2002-xg,
  author = {Fuchs, Eran and Jaffe, Jules and Long, Richard and Azam,
Farooq}, title = {Thin laser light sheet microscope for microbial oceanography}, journal = {Opt. Express}, publisher = {Optical Society of America}, year = {2002}, volume = {10}, number = {2}, pages = {145--154}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19424342}, doi = {http://dx.doi.org/10.1364/OE.10.000145} }
Galland, R., Grenci, G., Aravind, A., Viasnoff, V., Studer, V. and Sibarita, J.-B. 3D high- and super-resolution imaging using single-objective
SPIM
2015 Nat. Methods
Vol. 12(7), pp. 641-644 
article DOI URL 
Abstract: Single-objective selective-plane illumination microscopy
(soSPIM) is achieved with micromirrored cavities combined with
a laser beam-steering unit installed on a standard inverted
microscope. The illumination and detection are done through
the same objective. soSPIM can be used with standard sample
preparations and features high background rejection and
efficient photon collection, allowing for 3D
single-molecule-based super-resolution imaging of whole cells
or cell aggregates. Using larger mirrors enabled us to broaden
the capabilities of our system to image Drosophila embryos.
BibTeX:
@article{Galland2015-qm,
  author = {Galland, Remi and Grenci, Gianluca and Aravind, Ajay and
Viasnoff, Virgile and Studer, Vincent and Sibarita,
Jean-Baptiste}, title = {3D high- and super-resolution imaging using single-objective
SPIM}, journal = {Nat. Methods}, year = {2015}, volume = {12}, number = {7}, pages = {641--644}, url = {http://dx.doi.org/10.1038/nmeth.3402}, doi = {http://dx.doi.org/10.1038/nmeth.3402} }
Gao, L. Extend the field of view of selective plan illumination
microscopy by tiling the excitation light sheet
2015 Opt. Express
Vol. 23(5), pp. 6102-6111 
article DOI URL 
Abstract: Selective Plane Illumination Microscopy (SPIM) is attractive for
its ability to acquire 3D images with high 3D spatial
resolution, good optical sectioning capability and high imaging
speed. However, tradeoffs have to be made when a large field of
view (FOV) is required, results in lower axial resolution or
worse optical sectioning capability. Here, we present a novel
method for 3D imaging by SPIM that is capable to maintain its
high 3D spatial resolution and good optical sectioning
capability within a large FOV. Instead of trying to generate a
large and uniformly thick excitation light sheet, the method
tiles a relative small light sheet quickly to multiple positions
within the image plane by defocusing the excitation beam used to
create the light sheet, and takes one additional image at each
position, so that a large FOV can be imaged by repeating this
process and stitching all images together. By implementing this
method, light sheets with thin thickness and good excitation
light confinement can be used for SPIM imaging with slightly
compromised imaging speed. The method was investigated through
both numerical simulation and experiments, and the imaging
performance was demonstrated by imaging fluorescent particles
embedded in agarose gel and live C. elegans embryos.
BibTeX:
@article{Gao2015-qm,
  author = {Gao, Liang},
  title = {Extend the field of view of selective plan illumination
microscopy by tiling the excitation light sheet}, journal = {Opt. Express}, publisher = {Optical Society of America}, year = {2015}, volume = {23}, number = {5}, pages = {6102--6111}, url = {http://dx.doi.org/10.1364/OE.23.006102}, doi = {http://dx.doi.org/10.1364/OE.23.006102} }
Gao, L. Optimization of the excitation light sheet in selective plane
illumination microscopy
2015 Biomed. Opt. Express
Vol. 6(3), pp. 881-890 
article DOI URL 
Abstract: Selective plane illumination microscopy (SPIM) allows rapid 3D
live fluorescence imaging on biological specimens with high 3D
spatial resolution, good optical sectioning capability and
minimal photobleaching and phototoxic effect. SPIM gains its
advantage by confining the excitation light near the detection
focal plane, and its performance is determined by the ability
to create a thin, large and uniform excitation light sheet.
Several methods have been developed to create such an
excitation light sheet for SPIM. However, each method has its
own strengths and weaknesses, and tradeoffs must be made among
different aspects in SPIM imaging. In this work, we present a
strategy to select the excitation light sheet among the latest
SPIM techniques, and to optimize its geometry based on spatial
resolution, field of view, optical sectioning capability, and
the sample to be imaged. Besides the light sheets discussed in
this work, the proposed strategy is also applicable to
estimate the SPIM performance using other excitation light
sheets.
BibTeX:
@article{Gao2015-sc,
  author = {Gao, Liang},
  title = {Optimization of the excitation light sheet in selective plane
illumination microscopy}, journal = {Biomed. Opt. Express}, publisher = {Optical Society of America}, year = {2015}, volume = {6}, number = {3}, pages = {881--890}, url = {http://dx.doi.org/10.1364/BOE.6.000881}, doi = {http://dx.doi.org/10.1364/BOE.6.000881} }
Gao, L., Shao, L., Chen, B.-C. and Betzig, E. 3D live fluorescence imaging of cellular dynamics using
Bessel beam plane illumination microscopy
2014 Nat. Protoc.
Vol. 9(5), pp. 1083-1101 
article DOI URL 
Abstract: 3D live imaging is important for a better understanding of
biological processes, but it is challenging with current
techniques such as spinning-disk confocal microscopy. Bessel
beam plane illumination microscopy allows high-speed 3D live
fluorescence imaging of living cellular and multicellular
specimens with nearly isotropic spatial resolution, low
photobleaching and low photodamage. Unlike conventional
fluorescence imaging techniques that usually have a unique
operation mode, Bessel plane illumination has several modes
that offer different performance with different imaging
metrics. To achieve optimal results from this technique, the
appropriate operation mode needs to be selected and the
experimental setting must be optimized for the specific
application and associated sample properties. Here we explain
the fundamental working principles of this technique, discuss
the pros and cons of each operational mode and show through
examples how to optimize experimental parameters. We also
describe the procedures needed to construct, align and operate
a Bessel beam plane illumination microscope by using our
previously reported system as an example, and we list the
necessary equipment to build such a microscope. Assuming all
components are readily available, it would take a person
skilled in optical instrumentation 1 month to assemble and
operate a microscope according to this protocol.
BibTeX:
@article{Gao2014-ek,
  author = {Gao, Liang and Shao, Lin and Chen, Bi-Chang and Betzig, Eric},
  title = {3D live fluorescence imaging of cellular dynamics using
Bessel beam plane illumination microscopy}, journal = {Nat. Protoc.}, year = {2014}, volume = {9}, number = {5}, pages = {1083--1101}, url = {http://dx.doi.org/10.1038/nprot.2014.087}, doi = {http://dx.doi.org/10.1038/nprot.2014.087} }
Gao, L., Shao, L., Higgins, C.D., Poulton, J.S., Peifer, M., Davidson, M.W., Wu, X., Goldstein, B. and Betzig, E. Noninvasive imaging beyond the diffraction limit of 3D
dynamics in thickly fluorescent specimens
2012 Cell
Vol. 151(6), pp. 1370-1385 
article DOI URL 
Abstract: Optical imaging of the dynamics of living specimens involves
tradeoffs between spatial resolution, temporal resolution, and
phototoxicity, made more difficult in three dimensions. Here,
however, we report that rapid three-dimensional (3D) dynamics
can be studied beyond the diffraction limit in thick or
densely fluorescent living specimens over many time points by
combining ultrathin planar illumination produced by scanned
Bessel beams with super-resolution structured illumination
microscopy. We demonstrate in vivo karyotyping of chromosomes
during mitosis and identify different dynamics for the actin
cytoskeleton at the dorsal and ventral surfaces of
fibroblasts. Compared to spinning disk confocal microscopy, we
demonstrate substantially reduced photodamage when imaging
rapid morphological changes in D. discoideum cells, as well as
improved contrast and resolution at depth within developing C.
elegans embryos. Bessel beam structured plane illumination
thus promises new insights into complex biological phenomena
that require 4D subcellular spatiotemporal detail in either a
single or multicellular context.
BibTeX:
@article{Gao2012-xi,
  author = {Gao, Liang and Shao, Lin and Higgins, Christopher D and
Poulton, John S and Peifer, Mark and Davidson, Michael W and
Wu, Xufeng and Goldstein, Bob and Betzig, Eric}, title = {Noninvasive imaging beyond the diffraction limit of 3D
dynamics in thickly fluorescent specimens}, journal = {Cell}, year = {2012}, volume = {151}, number = {6}, pages = {1370--1385}, url = {http://dx.doi.org/10.1016/j.cell.2012.10.008}, doi = {http://dx.doi.org/10.1016/j.cell.2012.10.008} }
Gao, L., Zhu, L., Li, C. and Wang, L.V. Nonlinear light-sheet fluorescence microscopy by
photobleaching imprinting
2014 J. R. Soc. Interface
Vol. 11(93), pp. 20130851 
article DOI URL 
Abstract: We present a nonlinear light-sheet fluorescence microscopy
(LSFM) scheme based on photobleaching imprinting. By measuring
photobleaching-induced fluorescence decay, our method
simultaneously achieves a large imaging field of view and a
thin optical section. Furthermore, the scattered-light-induced
background is significantly reduced, considerably improving
image contrast. Our method is expected to expand the
application field of LSFM into the optical quasi-ballistic
regime, enabling studies on non-transparent biological
samples.
BibTeX:
@article{Gao2014-ca,
  author = {Gao, Liang and Zhu, Liren and Li, Chiye and Wang, Lihong V},
  title = {Nonlinear light-sheet fluorescence microscopy by
photobleaching imprinting}, journal = {J. R. Soc. Interface}, year = {2014}, volume = {11}, number = {93}, pages = {20130851}, url = {http://dx.doi.org/10.1098/rsif.2013.0851}, doi = {http://dx.doi.org/10.1098/rsif.2013.0851} }
Gebhardt, J.C.M., Suter, D.M., Roy, R., Zhao, Z.W., Chapman, A.R., Basu, S., Maniatis, T. and Xie, X.S. Single-molecule imaging of transcription factor binding to
DNA in live mammalian cells
2013 Nat. Methods
Vol. 10(5), pp. 421-426 
article DOI URL 
Abstract: Imaging single fluorescent proteins in living mammalian cells
is challenged by out-of-focus fluorescence excitation. To
reduce out-of-focus fluorescence we developed reflected
light-sheet microscopy (RLSM), a fluorescence microscopy
method allowing selective plane illumination throughout the
nuclei of living mammalian cells. A thin light sheet parallel
to the imaging plane and close to the sample surface is
generated by reflecting an elliptical laser beam incident from
the top by 90° with a small mirror. The thin light sheet
allows for an increased signal-to-background ratio superior to
that in previous illumination schemes and enables imaging of
single fluorescent proteins with up to 100-Hz time resolution.
We demonstrated the single-molecule sensitivity of RLSM by
measuring the DNA-bound fraction of glucocorticoid receptor
(GR) and determining the residence times on DNA of various
oligomerization states and mutants of GR and estrogen
receptor-$ (ER), which permitted us to resolve
different modes of DNA binding of GR. We demonstrated
two-color single-molecule imaging by observing the
spatiotemporal colocalization of two different protein pairs.
Our single-molecule measurements and statistical analysis
revealed dynamic properties of transcription factors.
BibTeX:
@article{Gebhardt2013-vy,
  author = {Gebhardt, J Christof M and Suter, David M and Roy, Rahul and
Zhao, Ziqing W and Chapman, Alec R and Basu, Srinjan and
Maniatis, Tom and Xie, X Sunney}, title = {Single-molecule imaging of transcription factor binding to
DNA in live mammalian cells}, journal = {Nat. Methods}, year = {2013}, volume = {10}, number = {5}, pages = {421--426}, url = {http://dx.doi.org/10.1038/nmeth.2411}, doi = {http://dx.doi.org/10.1038/nmeth.2411} }
Greger, K. Dynamic 3D Fluorescence Lifetime Imaging using Selective Plane
Illumination Microscopy (SPIM)
2008 FOM 2008, pp. 1-1  inproceedings  
BibTeX:
@inproceedings{Greger2008-de,
  author = {Greger, Klaus},
  title = {Dynamic 3D Fluorescence Lifetime Imaging using Selective Plane
Illumination Microscopy (SPIM)}, booktitle = {FOM 2008}, year = {2008}, pages = {1--1} }
Greger, K., Neetz, M.J., Reynaud, E.G. and Stelzer, E.H.K. Three-dimensional Fluorescence Lifetime Imaging with a Single
Plane Illumination Microscope provides an improved signal to
noise ratio
2011 Opt. Express
Vol. 19(21), pp. 20743-20750 
article DOI URL 
Abstract: We designed a widefield frequency domain Fluorescence Lifetime
Imaging Microscopy (FLIM)setup, which is based on a Single
Plane Illumination Microscope (SPIM). A SPIM provides an
inherent optical sectioning capability and reduces
photobleaching compared to conventional widefield and confocal
fluorescence microscopes. The lifetime precision of the FLIM
was characterized with Rhodamine 6G solutions of different
quencher concentrations [KI]. We demonstrate the high spatial
resolution of the SPIM-FLIM combination in the intensity
domain as well as in the lifetime domain with latex bead
samples and multiple recordings of three-dimensional live
Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the
bleaching rate after 600 images have been recorded is below
5
BibTeX:
@article{Greger2011-ft,
  author = {Greger, Klaus and Neetz, Manuel J and Reynaud, Emmanuel G and
Stelzer, Ernst H K}, title = {Three-dimensional Fluorescence Lifetime Imaging with a Single
Plane Illumination Microscope provides an improved signal to
noise ratio}, journal = {Opt. Express}, year = {2011}, volume = {19}, number = {21}, pages = {20743--20750}, url = {http://dx.doi.org/10.1364/OE.19.020743}, doi = {http://dx.doi.org/10.1364/OE.19.020743} }
Greger, K., Swoger, J. and Stelzer, E.H.K. Basic building units and properties of a fluorescence single
plane illumination microscope
2007 Rev. Sci. Instrum.
Vol. 78(2), pp. 023705 
article URL 
Abstract: The critical issue of all fluorescence microscopes is the
efficient use of the fluorophores, i.e., to detect as many
photons from the excited fluorophores as possible, as well as
to excite only the fluorophores that are in focus. This issue
is addressed in EMBL's implementation of a light sheet based
microscope [single plane illumination microscope (SPIM)],
which illuminates only the fluorophores in the focal plane of
the detection objective lens. The light sheet is a beam that
is collimated in one and focused in the other direction. Since
no fluorophores are excited outside the detectors' focal
plane, the method also provides intrinsic optical sectioning.
The total number of observable time points can be improved by
several orders of magnitude when compared to a confocal
fluorescence microscope. The actual improvement factor depends
on the number of planes acquired and required to achieve a
certain signal to noise ratio. A SPIM consists of five basic
units, which address (1) light detection, (2) illumination of
the specimen, (3) generation of an appropriate beam of light,
(4) translation and rotation of the specimen, and finally (5)
control of different mechanical and electronic parts, data
collection, and postprocessing of the data. We first describe
the basic building units of EMBL's SPIM and its most relevant
properties. We then cover the basic principles underlying this
instrument and its unique properties such as the efficient
usage of the fluorophores, the reduced photo toxic effects,
the true optical sectioning capability, and the excellent
axial resolution. We also discuss how an isotropic resolution
can be achieved. The optical setup, the control hardware, and
the control scheme are explained in detail. We also describe
some less obvious refinements of the basic setup that result
in an improved performance. The properties of the instrument
are demonstrated by images of biological samples that were
imaged with one of EMBL's SPIMs.
BibTeX:
@article{Greger2007-nf,
  author = {Greger, K and Swoger, J and Stelzer, E H K},
  title = {Basic building units and properties of a fluorescence single
plane illumination microscope}, journal = {Rev. Sci. Instrum.}, year = {2007}, volume = {78}, number = {2}, pages = {023705}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17578115} }
Gualda, E., Moreno, N., Tomancak, P. and Martins, G.G. Going`` open'' with Mesoscopy: a new dimension on multi-view
imaging
2014 Protoplasma
Vol. 251(2), pp. 363-372 
article URL 
Abstract: Abstract OpenSPIM and OpenSpinMicroscopy emerged as open access
platforms for Light Sheet and Optical Projection Imaging , often
called as optical mesoscopy techniques. Both projects can be
easily reproduced using comprehensive online instructions that
should ...
BibTeX:
@article{Gualda2014-uc,
  author = {Gualda, Emilio and Moreno, Nuno and Tomancak, Pavel and Martins,
Gabriel G}, title = {Going`` open'' with Mesoscopy: a new dimension on multi-view
imaging}, journal = {Protoplasma}, publisher = {Springer}, year = {2014}, volume = {251}, number = {2}, pages = {363--372}, url = {http://link.springer.com/article/10.1007/s00709-013-0599-3} }
Gualda, E.J., Sim ao, D., Pinto, C., Alves, P.M. and Brito, C. Imaging of human differentiated 3D neural aggregates using
light sheet fluorescence microscopy
2014 Front. Cell. Neurosci.
Vol. 8, pp. 221 
article DOI URL 
Abstract: The development of three dimensional (3D) cell cultures
represents a big step for the better understanding of cell
behavior and disease in a more natural like environment,
providing not only single but multiple cell type interactions
in a complex 3D matrix, highly resembling physiological
conditions. Light sheet fluorescence microscopy (LSFM) is
becoming an excellent tool for fast imaging of such 3D
biological structures. We demonstrate the potential of this
technique for the imaging of human differentiated 3D neural
aggregates in fixed and live samples, namely calcium imaging
and cell death processes, showing the power of imaging
modality compared with traditional microscopy. The combination
of light sheet microscopy and 3D neural cultures will open the
door to more challenging experiments involving drug testing at
large scale as well as a better understanding of relevant
biological processes in a more realistic environment.
BibTeX:
@article{Gualda2014-ur,
  author = {Gualda, Emilio J and Simão, Daniel and Pinto, Catarina and
Alves, Paula M and Brito, Catarina}, title = {Imaging of human differentiated 3D neural aggregates using
light sheet fluorescence microscopy}, journal = {Front. Cell. Neurosci.}, publisher = {Frontiers}, year = {2014}, volume = {8}, pages = {221}, url = {http://dx.doi.org/10.3389/fncel.2014.00221}, doi = {http://dx.doi.org/10.3389/fncel.2014.00221} }
Gualda, E.J., Vale, T., Almada, P., Feijó, J.A., Martins, G.G. and Moreno, N. OpenSpinMicroscopy: an open-source integrated microscopy
platform
2013 Nat. Methods
Vol. 10(7), pp. 599-600 
article DOI URL 
BibTeX:
@article{Gualda2013-de,
  author = {Gualda, Emilio J and Vale, Tiago and Almada, Pedro and Feijó,
José A and Martins, Gabriel G and Moreno, Nuno}, title = {OpenSpinMicroscopy: an open-source integrated microscopy
platform}, journal = {Nat. Methods}, year = {2013}, volume = {10}, number = {7}, pages = {599--600}, url = {http://dx.doi.org/10.1038/nmeth.2508}, doi = {http://dx.doi.org/10.1038/nmeth.2508} }
Hägerling, R., Pollmann, C., Andreas, M., Schmidt, C., Nurmi, H., Adams, R.H., Alitalo, K., Andresen, V., Schulte-Merker, S. and Kiefer, F. A novel multistep mechanism for initial lymphangiogenesis in
mouse embryos based on ultramicroscopy
2013 EMBO J.
Vol. 32(5), pp. 629-644 
article DOI URL 
Abstract: During mammalian development, a subpopulation of endothelial
cells in the cardinal vein (CV) expresses lymphatic-specific
genes and subsequently develops into the first lymphatic
structures, collectively termed as lymph sacs. Budding,
sprouting and ballooning of lymphatic endothelial cells (LECs)
have been proposed to underlie the emergence of LECs from the
CV, but the exact mechanisms of lymph vessel formation remain
poorly understood. Applying selective plane illumination-based
ultramicroscopy to entire wholemount-immunostained mouse
embryos, we visualized the complete developing vascular system
with cellular resolution. Here, we report emergence of the
earliest detectable LECs as strings of loosely connected cells
between the CV and superficial venous plexus. Subsequent
aggregation of LECs resulted in formation of two distinct,
previously unidentified lymphatic structures, the dorsal
peripheral longitudinal lymphatic vessel (PLLV) and the
ventral primordial thoracic duct (pTD), which at later stages
formed a direct contact with the CV. Providing new insights
into their function, we found vascular endothelial growth
factor C (VEGF-C) and the matrix component CCBE1 indispensable
for LEC budding and migration. Altogether, we present a
significantly more detailed view and novel model of early
lymphatic development.
BibTeX:
@article{Hagerling2013-rd,
  author = {Hägerling, René and Pollmann, Cathrin and Andreas,
Martin and Schmidt, Christian and Nurmi, Harri and Adams, Ralf
H and Alitalo, Kari and Andresen, Volker and Schulte-Merker,
Stefan and Kiefer, Friedemann}, title = {A novel multistep mechanism for initial lymphangiogenesis in
mouse embryos based on ultramicroscopy}, journal = {EMBO J.}, year = {2013}, volume = {32}, number = {5}, pages = {629--644}, url = {http://dx.doi.org/10.1038/emboj.2012.340}, doi = {http://dx.doi.org/10.1038/emboj.2012.340} }
Höckendorf, B., Lavis, L.D. and Keller, P.J. Making biology transparent 2014 Nat. Biotechnol.
Vol. 32(11), pp. 1104-1105 
article DOI URL 
BibTeX:
@article{Hockendorf2014-ls,
  author = {Höckendorf, Burkhard and Lavis, Luke D and Keller,
Philipp J}, title = {Making biology transparent}, journal = {Nat. Biotechnol.}, year = {2014}, volume = {32}, number = {11}, pages = {1104--1105}, url = {http://dx.doi.org/10.1038/nbt.3061}, doi = {http://dx.doi.org/10.1038/nbt.3061} }
Höckendorf, B., Thumberger, T. and Wittbrodt, J. Quantitative analysis of embryogenesis: a perspective for
light sheet microscopy
2012 Dev. Cell
Vol. 23(6), pp. 1111-1120 
article DOI URL 
Abstract: It is a challenge in developmental biology to understand how
an embryo's genes, proteins, and cells function and interact
to govern morphogenesis, cell fate specification, and
patterning. These processes span very different spatial and
temporal scales. Despite much progress, simultaneous
observation of such vastly differing scales has been beyond
the scope of conventional microscopy. Light sheet microscopy
fills this gap and is increasingly used for long-term,
high-speed recordings of large specimens with high contrast
and up to subcellular spatial resolution. We provide an
overview of applications of light sheet microscopy in
developmental biology and discuss future perspectives in this
field.
BibTeX:
@article{Hockendorf2012-by,
  author = {Höckendorf, Burkhard and Thumberger, Thomas and
Wittbrodt, Joachim}, title = {Quantitative analysis of embryogenesis: a perspective for
light sheet microscopy}, journal = {Dev. Cell}, year = {2012}, volume = {23}, number = {6}, pages = {1111--1120}, url = {http://dx.doi.org/10.1016/j.devcel.2012.10.008}, doi = {http://dx.doi.org/10.1016/j.devcel.2012.10.008} }
Hammers, M.D., Taormina, M.J., Cerda, M.M., Montoya, L.A., Seidenkranz, D.T., Parthasarathy, R. and Pluth, M.D. A Bright Fluorescent Probe for H2S Enables
Analyte-Responsive, 3D Imaging in Live Zebrafish Using
Light Sheet Fluorescence Microscopy
2015 J. Am. Chem. Soc.
Vol. 137(32), pp. 10216-10223 
article DOI URL 
Abstract: Hydrogen sulfide (H2S) is a critical gaseous signaling
molecule emerging at the center of a rich field of chemical
and biological research. As our understanding of the
complexity of physiological H2S in signaling pathways evolves,
advanced chemical and technological investigative tools are
required to make sense of this interconnectivity. Toward this
goal, we have developed an azide-functionalized O-methylrhodol
fluorophore, MeRho-Az, which exhibits a rapid >1000-fold
fluorescence response when treated with H2S, is selective for
H2S over other biological analytes, and has a detection limit
of 86 nM. Additionally, the MeRho-Az scaffold is less
susceptible to photoactivation than other commonly used
azide-based systems, increasing its potential application in
imaging experiments. To demonstrate the efficacy of this probe
for H2S detection, we demonstrate the ability of MeRho-Az to
detect differences in H2S levels in C6 cells and those treated
with AOAA, a common inhibitor of enzymatic H2S synthesis.
Expanding the use of MeRho-Az to complex and heterogeneous
biological settings, we used MeRho-Az in combination with
light sheet fluorescence microscopy (LSFM) to visualize H2S in
the intestinal tract of live zebrafish. This application
provides the first demonstration of analyte-responsive 3D
imaging with LSFM, highlighting the utility of combining new
probes and live imaging methods for investigating chemical
signaling in complex multicellular systems.
BibTeX:
@article{Hammers2015-xj,
  author = {Hammers, Matthew D and Taormina, Michael J and Cerda, Matthew
M and Montoya, Leticia A and Seidenkranz, Daniel T and
Parthasarathy, Raghuveer and Pluth, Michael D}, title = {A Bright Fluorescent Probe for H2S Enables
Analyte-Responsive, 3D Imaging in Live Zebrafish Using
Light Sheet Fluorescence Microscopy}, journal = {J. Am. Chem. Soc.}, year = {2015}, volume = {137}, number = {32}, pages = {10216--10223}, url = {http://dx.doi.org/10.1021/jacs.5b04196}, doi = {http://dx.doi.org/10.1021/jacs.5b04196} }
Hedde, P.N. and Gratton, E. Active focus stabilization for upright selective plane
illumination microscopy
2015 Opt. Express
Vol. 23(11), pp. 14707-14714 
article DOI URL 
Abstract: Due to its sectioning capability, large field of view, and
minimal light exposure, selective plane illumination microscopy
has become the preferred choice for 3D time lapse imaging. Single
cells in a dish can be conveniently imaged using an
upright/inverted configuration. However, for measurements on long
time scales (hours to days), mechanical drift is a problem;
especially for studies of mammalian cells that typically require
heating to 37°C which causes a thermal gradient across the
instrument. Since the light sheet diverges towards the edges of
the field of view, such a drift leads to a decrease in axial
resolution over time. Or, even worse, the specimen could move out
of the imaging volume. Here, we present a simple, cost-effective
way to stabilize the axial position using the microscope camera
to track the sample position. Thereby, sample loss is prevented
and an optimal axial resolution is maintained by keeping the
sample at the position where the light sheet is at its thinnest.
We demonstrate the virtue of our approach by measurements of the
light sheet thickness and 3D time lapse imaging of a cell
monolayer at physiological conditions.
BibTeX:
@article{Hedde2015-lq,
  author = {Hedde, Per Niklas and Gratton, Enrico},
  title = {Active focus stabilization for upright selective plane
illumination microscopy}, journal = {Opt. Express}, year = {2015}, volume = {23}, number = {11}, pages = {14707--14714}, url = {http://dx.doi.org/10.1364/OE.23.014707}, doi = {http://dx.doi.org/10.1364/OE.23.014707} }
Heintzmann, R. and Cremer, C. Axial tomographic confocal fluorescence microscopy 2002 J. Microsc.
Vol. 206(Pt 1), pp. 7-23 
article URL 
Abstract: By physical rotation of the sample, axial tomography enables
the acquisition of otherwise inaccessible spatial information
from an object. In combination with confocal microscopy, the
method can fundamentally improve the effective
three-dimensional (3D) resolution. In this report we present a
novel method for high resolution reconstruction of confocal
axial tomographic data. The method automatically determines
the relative angles of rotation, aligns the data from
different rotational views and reconstructs a single high
resolution 3D dataset. The reconstruction makes use of a known
point spread function and is based on an unconstrained maximum
likelihood deconvolution performed simultaneously from
multiple (in our case three) angular views. It was applied to
simulated as well as to experimental confocal datasets. The
gain in resolution was quantified and the effect of choice of
overrelaxation factors on the speed of convergence was
investigated. A clearly improved 3D resolution was obtained by
axial tomography together with reconstruction as compared with
reconstruction of confocal data from only a single angular
view.
BibTeX:
@article{Heintzmann2002-dt,
  author = {Heintzmann, R and Cremer, C},
  title = {Axial tomographic confocal fluorescence microscopy},
  journal = {J. Microsc.},
  year = {2002},
  volume = {206},
  number = {Pt 1},
  pages = {7--23},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/12000559}
}
Heintzmann, R., Kreth, G. and Cremer, C. Reconstruction of axial tomographic high resolution data from
confocal fluorescence microscopy: a method for improving 3D
FISH images
2000 Anal. Cell. Pathol.
Vol. 20(1), pp. 7-15 
article URL 
Abstract: Fluorescent confocal laser scanning microscopy allows an
improved imaging of microscopic objects in three dimensions.
However, the resolution along the axial direction is three
times worse than the resolution in lateral directions. A
method to overcome this axial limitation is tilting the object
under the microscope, in a way that the direction of the
optical axis points into different directions relative to the
sample. A new technique for a simultaneous reconstruction from
a number of such axial tomographic confocal data sets was
developed and used for high resolution reconstruction of
3D-data both from experimental and virtual microscopic data
sets. The reconstructed images have a highly improved 3D
resolution, which is comparable to the lateral resolution of a
single deconvolved data set. Axial tomographic imaging in
combination with simultaneous data reconstruction also opens
the possibility for a more precise quantification of 3D data.
The color images of this publication can be accessed from
http://www.esacp.org/acp/2000/20-1/heintzmann.++ +htm. At this
web address an interactive 3D viewer is additionally provided
for browsing the 3D data. This java applet displays three
orthogonal slices of the data set which are dynamically
updated by user mouse clicks or keystrokes.
BibTeX:
@article{Heintzmann2000-vq,
  author = {Heintzmann, R and Kreth, G and Cremer, C},
  title = {Reconstruction of axial tomographic high resolution data from
confocal fluorescence microscopy: a method for improving 3D
FISH images}, journal = {Anal. Cell. Pathol.}, year = {2000}, volume = {20}, number = {1}, pages = {7--15}, url = {http://www.ncbi.nlm.nih.gov/pubmed/11007433} }
Herbert, S.P., Huisken, J., Kim, T.N., Feldman, M.E., Houseman, B.T., Wang, R.A., Shokat, K.M. and Stainier, D.Y.R. Arterial-venous segregation by selective cell sprouting: an
alternative mode of blood vessel formation
2009 Science
Vol. 326(5950), pp. 294-298 
article DOI URL 
Abstract: Blood vessels form de novo (vasculogenesis) or upon sprouting
of capillaries from preexisting vessels (angiogenesis). With
high-resolution imaging of zebrafish vascular development, we
uncovered a third mode of blood vessel formation whereby the
first embryonic artery and vein, two unconnected blood
vessels, arise from a common precursor vessel. The first
embryonic vein formed by selective sprouting of progenitor
cells from the precursor vessel, followed by vessel
segregation. These processes were regulated by the ligand
EphrinB2 and its receptor EphB4, which are expressed in
arterial-fated and venous-fated progenitors, respectively, and
interact to orient the direction of progenitor migration.
Thus, directional control of progenitor migration drives
arterial-venous segregation and generation of separate
parallel vessels from a single precursor vessel, a process
essential for vascular development.
BibTeX:
@article{Herbert2009-zc,
  author = {Herbert, Shane P and Huisken, Jan and Kim, Tyson N and
Feldman, Morri E and Houseman, Benjamin T and Wang, Rong A and
Shokat, Kevan M and Stainier, Didier Y R}, title = {Arterial-venous segregation by selective cell sprouting: an
alternative mode of blood vessel formation}, journal = {Science}, year = {2009}, volume = {326}, number = {5950}, pages = {294--298}, url = {http://dx.doi.org/10.1126/science.1178577}, doi = {http://dx.doi.org/10.1126/science.1178577} }
Herzog, W., Müller, K., Huisken, J. and Stainier, D.Y.R. Genetic evidence for a noncanonical function of seryl-tRNA
synthetase in vascular development
2009 Circ. Res.
Vol. 104(11), pp. 1260-1266 
article DOI URL 
Abstract: In a recent genetic screen, we identified mutations in genes
important for vascular development and maintenance in
zebrafish (Jin et al. Dev Biol. 2007;307:29-42). Mutations
[corrected] at the adrasteia (adr) locus cause a pronounced
dilatation of the aortic arch vessels as well as aberrant
patterning of the hindbrain capillaries and, to a lesser
extent, intersomitic vessels. This dilatation of the aortic
arch vessels does not appear to be caused by increased cell
proliferation but is dependent on vascular endothelial growth
factor (Vegf) signaling. By positional cloning, we isolated
seryl-tRNA synthetase (sars) as the gene affected by the adr
mutations. Small interfering RNA knockdown experiments in
human umbilical vein endothelial cell cultures indicate that
SARS also regulates endothelial sprouting. These analyses of
zebrafish and human endothelial cells reveal a new
noncanonical function of Sars in endothelial development.
BibTeX:
@article{Herzog2009-zr,
  author = {Herzog, Wiebke and Müller, Katja and Huisken, Jan and
Stainier, Didier Y R}, title = {Genetic evidence for a noncanonical function of seryl-tRNA
synthetase in vascular development}, journal = {Circ. Res.}, year = {2009}, volume = {104}, number = {11}, pages = {1260--1266}, url = {http://dx.doi.org/10.1161/CIRCRESAHA.108.191718}, doi = {http://dx.doi.org/10.1161/CIRCRESAHA.108.191718} }
van 't Hoff, M., de Sars, V. and Oheim, M. A programmable light engine for quantitative single molecule
TIRF and HILO imaging
2008 Opt. Express
Vol. 16(22), pp. 18495-18504 
article URL 
Abstract: We report on a simple yet powerful implementation of
objective-type total internal reflection fluorescence (TIRF)
and highly inclined and laminated optical sheet (HILO, a type
of dark-field) illumination. Instead of focusing the
illuminating laser beam to a single spot close to the edge of
the microscope objective, we are scanning during the
acquisition of a fluorescence image the focused spot in a
circular orbit, thereby illuminating the sample from various
directions. We measure parameters relevant for quantitative
image analysis during fluorescence image acquisition by
capturing an image of the excitation light distribution in an
equivalent objective backfocal plane (BFP). Operating at scan
rates above 1 MHz, our programmable light engine allows
directional averaging by circular spinning the spot even for
sub-millisecond exposure times. We show that restoring the
symmetry of TIRF/HILO illumination reduces scattering and
produces an evenly lit field-of-view that affords on-line
analysis of evanescnt-field excited fluorescence without
pre-processing. Utilizing crossed acousto-optical deflectors,
our device generates arbitrary intensity profiles in BFP,
permitting variable-angle, multi-color illumination, or
objective lenses to be rapidly exchanged.
BibTeX:
@article{Van_t_Hoff2008-ab,
  author = {van 't Hoff, Marcel and de Sars, Vincent and Oheim, Martin},
  title = {A programmable light engine for quantitative single molecule
TIRF and HILO imaging}, journal = {Opt. Express}, year = {2008}, volume = {16}, number = {22}, pages = {18495--18504}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18958128} }
Hofman, R., Segenhout, J.M., Buytaert, J.A.N., Dirckx, J.J.J. and Wit, H.P. Morphology and function of Bast's valve: additional insight in
its functioning using 3D-reconstruction
2008 Eur. Arch. Otorhinolaryngol.
Vol. 265(2), pp. 153-157 
article DOI URL 
Abstract: The utriculo-endolymphatic valve was discovered by Bast in
1928. The function of Bast's valve is still unclear. By means
of orthogonal-plane fluorescence optical sectioning (OPFOS)
microscopy 3D-reconstructions of the valve and its surrounding
region are depicted. The shape of the duct at the utricular
side is that of a flattened funnel. In the direction of the
endolymphatic duct and sac this funnel runs into a very narrow
duct. The valve itself has a rigid 'arch-like' configuration.
The opposing thin, one cell-layer thick, utricular membrane is
highly compliant. We propose that opening and closure of the
valve occurs through movement of the flexible base/utricular
membrane away from and toward the relatively rigid valve lip.
BibTeX:
@article{Hofman2008-vp,
  author = {Hofman, R and Segenhout, J M and Buytaert, J A N and Dirckx, J
J J and Wit, H P}, title = {Morphology and function of Bast's valve: additional insight in
its functioning using 3D-reconstruction}, journal = {Eur. Arch. Otorhinolaryngol.}, year = {2008}, volume = {265}, number = {2}, pages = {153--157}, url = {http://dx.doi.org/10.1007/s00405-007-0424-8}, doi = {http://dx.doi.org/10.1007/s00405-007-0424-8} }
Hofman, R., Segenhout, J.M. and Wit, H.P. Three-dimensional reconstruction of the guinea pig inner ear,
comparison of OPFOS and light microscopy, applications of
3D reconstruction
2009 J. Microsc.
Vol. 233(2), pp. 251-257 
article DOI URL 
Abstract: Three-dimensional (3D) reconstruction of anatomical structures
can give additional insight into the morphology and function
of these structures. We compare 3D reconstructions of the
guinea pig inner ear, using light microscopy and orthogonal
plane fluorescence optical sectioning microscopy. Applications
of 3D reconstruction of the inner ear are further explored.
For each method two bullas were prepared for 3D
reconstruction. Both methods are explained. In general, the 3D
reconstructions using orthogonal plane fluorescence optical
sectioning microscopy are superior to light microscopy. The
exact spiral shape of the cochlea could be reconstructed using
orthogonal plane fluorescence optical sectioning microscopy
and the length of the basilar membrane measured. When a
resolution of 20 microm is sufficient, orthogonal plane
fluorescence optical sectioning microscopy is a superior
technique for 3D reconstruction of inner ear structures in
animals.
BibTeX:
@article{Hofman2009-yb,
  author = {Hofman, R and Segenhout, J M and Wit, H P},
  title = {Three-dimensional reconstruction of the guinea pig inner ear,
comparison of OPFOS and light microscopy, applications of
3D reconstruction}, journal = {J. Microsc.}, year = {2009}, volume = {233}, number = {2}, pages = {251--257}, url = {http://dx.doi.org/10.1111/j.1365-2818.2009.03115.x}, doi = {http://dx.doi.org/10.1111/j.1365-2818.2009.03115.x} }
Holekamp, T.F., Turaga, D. and Holy, T.E. Fast three-dimensional fluorescence imaging of activity in
neural populations by objective-coupled planar illumination
microscopy
2008 Neuron
Vol. 57(5), pp. 661-672 
article DOI URL 
Abstract: Unraveling the functions of the diverse neural types in any
local circuit ultimately requires methods to record from most
or all of its cells simultaneously. One promising approach to
this goal is fluorescence imaging, but existing methods using
laser-scanning microscopy (LSM) are severely limited in their
ability to resolve rapid phenomena, like neuronal action
potentials, over wide fields. Here we present a microscope
that rapidly sections a three-dimensional volume using a thin
illumination sheet whose position is rigidly coupled to the
objective and aligned with its focal plane. We demonstrate
that this approach allows exceptionally low-noise imaging of
large neuronal populations at pixel rates at least 100-fold
higher than with LSM. Using this microscope, we studied the
pheromone-sensing neurons of the mouse vomeronasal organ and
found that responses to dilute urine are largely or
exclusively restricted to cells in the apical layer, the
location of V1r-family-expressing neurons.
BibTeX:
@article{Holekamp2008-ls,
  author = {Holekamp, Terrence F and Turaga, Diwakar and Holy, Timothy E},
  title = {Fast three-dimensional fluorescence imaging of activity in
neural populations by objective-coupled planar illumination
microscopy}, journal = {Neuron}, year = {2008}, volume = {57}, number = {5}, pages = {661--672}, url = {http://dx.doi.org/10.1016/j.neuron.2008.01.011}, doi = {http://dx.doi.org/10.1016/j.neuron.2008.01.011} }
Holy, T.E. Calcium imaging in populations of olfactory neurons by planar
illumination microscopy
2014 Cold Spring Harb. Protoc.
Vol. 2014(3), pp. 317-323 
article DOI URL 
Abstract: Neurons in the olfactory system display extraordinary functional
diversity, which at the level of sensory neurons arises from the
expression of one out of several hundred distinct receptor
types. To cope with this diversity, one approach is to use
techniques that can record sensory responses from many neurons
simultaneously. We have developed a form of light-sheet
microscopy, called objective-coupled planar illumination (OCPI)
microscopy, that is well suited to recording at high
signal-to-noise ratios from large neuronal populations. Because
OCPI microscopy illuminates the entire field simultaneously, it
allows fast imaging without compromising field of view. At
current camera speeds, pixels can be acquired more than 100-fold
faster than by point-scanning fluorescence microscopy. Here we
describe the theory, advantages, and practical implementation of
planar illumination and briefly discuss its application to
neuronal recording in the mouse vomeronasal organ. We also
provide a brief protocol, in which a mouse is pretreated with
dye for 1 wk to allow labeling of the sensory neurons before
stimulation and imaging.
BibTeX:
@article{Holy2014-zc,
  author = {Holy, Timothy E},
  title = {Calcium imaging in populations of olfactory neurons by planar
illumination microscopy}, journal = {Cold Spring Harb. Protoc.}, publisher = {cshprotocols.cshlp.org}, year = {2014}, volume = {2014}, number = {3}, pages = {317--323}, url = {http://dx.doi.org/10.1101/pdb.prot081174}, doi = {http://dx.doi.org/10.1101/pdb.prot081174} }
Hu, Y., Zhu, Q., Elkins, K., Tse, K., Li, Y., Fitzpatrick, J., Verma, I. and Cang, H. Light-sheet Bayesian microscopy enables deep-cell
super-resolution imaging of heterochromatin in live human
embryonic stem cells
2013 Optical Nanoscopy
Vol. 2(1), pp. 7 
misc DOI URL 
Abstract: BACKGROUND:Heterochromatin in the nucleus of human embryonic
cells plays an important role in the epigenetic regulation of
gene expression. The architecture of heterochromatin and its
dynamic organization remain elusive because of the lack of fast
and high-resolution deep-cell imaging tools. We enable this task
by advancing instrumental and algorithmic implementation of the
localization-based super-resolution technique.RESULTS:We present
light-sheet Bayesian super-resolution microscopy (LSBM). We adapt
light-sheet illumination for super-resolution imaging by using a
novel prism-coupled condenser design to illuminate a thin slice
of the nucleus with high signal-to-noise ratio. Coupled with a
Bayesian algorithm that resolves overlapping fluorophores from
high-density areas, we show, for the first time, nanoscopic
features of the heterochromatin structure in both fixed and live
human embryonic stem cells. The enhanced temporal resolution
allows capturing the dynamic change of heterochromatin with a
lateral resolution of 50-60 nm on a time scale of 2.3
s.CONCLUSION:Light-sheet Bayesian microscopy opens up broad new
possibilities of probing nanometer-scale nuclear structures and
real-time sub-cellular processes and other previously
difficult-to-access intracellular regions of living cells at the
single-molecule, and single cell level.
BibTeX:
@misc{Hu2013-ti,
  author = {Hu, Ying and Zhu, Quan and Elkins, Keri and Tse, Kevin and Li, Yu
and Fitzpatrick, James and Verma, Inder and Cang, Hu}, title = {Light-sheet Bayesian microscopy enables deep-cell
super-resolution imaging of heterochromatin in live human
embryonic stem cells}, journal = {Optical Nanoscopy}, year = {2013}, volume = {2}, number = {1}, pages = {7}, url = {http://www.optnano.com/content/2/1/7}, doi = {http://dx.doi.org/10.1186/2192-2853-2-7} }
Hu, Y.S., Zimmerley, M., Li, Y., Watters, R. and Cang, H. Single-molecule super-resolution light-sheet microscopy 2014 Chemphyschem
Vol. 15(4), pp. 577-586 
article DOI URL 
Abstract: Single-molecule super-resolution imaging is a new promising
tool for investigation of sub-cellular structures.
Concurrently, light-sheet microscopy, also known as selective
plane illumination microscopy (SPIM), has gained rapid favor
with the imaging community in developmental biology due to its
fast speed, high contrast, deep penetration, and low
phototoxicity. While nearly a dozen reviews thoroughly
describe the development of light-sheet microscopy and its
technological breakthroughs with a main focus on improving the
3D imaging speed of fish embryos, central nervous system, and
other tissues, few have addressed the potential of combining
light-sheet microscopy and localization-based super-resolution
imaging to achieve sub-diffraction-limited resolution.
Adapting light-sheet illumination for single-molecule imaging
presents unique challenges for instrumentation and
reconstruction algorithms. In this Minireview, we provide an
overview of the recent developments that address these
challenges. We compare different approaches in
super-resolution and light-sheet imaging, address advantages
and limitations in each approach, and outline future
directions of this emerging field.
BibTeX:
@article{Hu2014-hr,
  author = {Hu, Ying S and Zimmerley, Maxwell and Li, Yu and Watters,
Robin and Cang, Hu}, title = {Single-molecule super-resolution light-sheet microscopy}, journal = {Chemphyschem}, publisher = {Wiley Online Library}, year = {2014}, volume = {15}, number = {4}, pages = {577--586}, url = {http://dx.doi.org/10.1002/cphc.201300732}, doi = {http://dx.doi.org/10.1002/cphc.201300732} }
Huber, D., Keller, M. and Robert, D. 3D light scanning macrography 2001 J. Microsc.
Vol. 203(Pt 2), pp. 208-213 
article URL 
Abstract: The technique of 3D light scanning macrography permits the
non-invasive surface scanning of small specimens at
magnifications up to 200x. Obviating both the problem of
limited depth of field inherent to conventional close-up
macrophotography and the metallic coating required by scanning
electron microscopy, 3D light scanning macrography provides
three-dimensional digital images of intact specimens without
the loss of colour, texture and transparency information. This
newly developed technique offers a versatile, portable and
cost-efficient method for the non-invasive digital and
photographic documentation of small objects. Computer
controlled device operation and digital image acquisition
facilitate fast and accurate quantitative morphometric
investigations, and the technique offers a broad field of
research and educational applications in biological, medical
and materials sciences.
BibTeX:
@article{Huber2001-gy,
  author = {Huber, D and Keller, M and Robert, D},
  title = {3D light scanning macrography},
  journal = {J. Microsc.},
  year = {2001},
  volume = {203},
  number = {Pt 2},
  pages = {208--213},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/11489078}
}
Huisken, J. Slicing embryos gently with laser light sheets 2012 Bioessays
Vol. 34(5), pp. 406-411 
article DOI URL 
Abstract: Light sheet microscopy is an easy to implement and extremely
powerful alternative to established fluorescence imaging
techniques such as laser scanning confocal, multi-photon and
spinning disk microscopy. By illuminating the sample only with
a thin slice of light, photo-bleaching is reduced to a
minimum, making light sheet microscopy ideal for
non-destructive imaging of fragile samples over extended
periods of time. Millimeter-sized samples can be imaged
rapidly with high resolution and high depth penetration. A
large variety of instruments have been developed and optimized
for a number of different samples: Bessel beams form thin
light sheets for single cells, and selective plane
illumination microscopy (SPIM) offers multi-view acquisition
to image entire embryos with isotropic resolution. This review
explains how light sheet microscopy involves a conceptually
new microscope design and how it changes modern imaging in
biology.
BibTeX:
@article{Huisken2012-dt,
  author = {Huisken, Jan},
  title = {Slicing embryos gently with laser light sheets},
  journal = {Bioessays},
  year = {2012},
  volume = {34},
  number = {5},
  pages = {406--411},
  url = {http://dx.doi.org/10.1002/bies.201100120},
  doi = {http://dx.doi.org/10.1002/bies.201100120}
}
Huisken, J. and Stainier, D.Y.R. Selective plane illumination microscopy techniques in
developmental biology
2009 Development
Vol. 136(12), pp. 1963-1975 
article DOI URL 
Abstract: Selective plane illumination microscopy (SPIM) and other
fluorescence microscopy techniques in which a focused sheet of
light serves to illuminate the sample have become increasingly
popular in developmental studies. Fluorescence light-sheet
microscopy bridges the gap in image quality between
fluorescence stereomicroscopy and high-resolution imaging of
fixed tissue sections. In addition, high depth penetration,
low bleaching and high acquisition speeds make light-sheet
microscopy ideally suited for extended time-lapse experiments
in live embryos. This review compares the benefits and
challenges of light-sheet microscopy with established
fluorescence microscopy techniques such as confocal microscopy
and discusses the different implementations and applications
of this easily adaptable technology.
BibTeX:
@article{Huisken2009-iw,
  author = {Huisken, Jan and Stainier, Didier Y R},
  title = {Selective plane illumination microscopy techniques in
developmental biology}, journal = {Development}, year = {2009}, volume = {136}, number = {12}, pages = {1963--1975}, url = {http://dx.doi.org/10.1242/dev.022426}, doi = {http://dx.doi.org/10.1242/dev.022426} }
Huisken, J. and Stainier, D.Y.R. Even fluorescence excitation by multidirectional selective
plane illumination microscopy (mSPIM)
2007 Opt. Lett.
Vol. 32(17), pp. 2608-2610 
article URL 
Abstract: Multidirectional selective plane illumination microscopy
(mSPIM) reduces absorption and scattering artifacts and
provides an evenly illuminated focal plane. mSPIM solves two
common problems in light-sheet-based imaging techniques: The
shadowing in the excitation path due to absorption in the
specimen is eliminated by pivoting the light sheet; the spread
of the light sheet by scattering in the sample is compensated
by illuminating the sample consecutively from opposing
directions. The resulting two images are computationally fused
yielding a superior image. The effective light sheet is
thinner, and the axial resolution is increased by square root
2 over single-directional SPIM. The multidirectional
illumination proves essential in biological specimens such as
millimeter-sized embryos. The performance of mSPIM is
demonstrated by the imaging of live zebrafish embryos.
BibTeX:
@article{Huisken2007-zg,
  author = {Huisken, Jan and Stainier, Didier Y R},
  title = {Even fluorescence excitation by multidirectional selective
plane illumination microscopy (mSPIM)}, journal = {Opt. Lett.}, year = {2007}, volume = {32}, number = {17}, pages = {2608--2610}, url = {http://www.ncbi.nlm.nih.gov/pubmed/17767321} }
Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. and Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane
illumination microscopy
2004 Science
Vol. 305(5686), pp. 1007-1009 
article DOI URL 
Abstract: Large, living biological specimens present challenges to
existing optical imaging techniques because of their
absorptive and scattering properties. We developed selective
plane illumination microscopy (SPIM) to generate
multidimensional images of samples up to a few millimeters in
size. The system combines two-dimensional illumination with
orthogonal camera-based detection to achieve high-resolution,
optically sectioned imaging throughout the sample, with
minimal photodamage and at speeds capable of capturing
transient biological phenomena. We used SPIM to visualize all
muscles in vivo in the transgenic Medaka line Arnie, which
expresses green fluorescent protein in muscle tissue. We also
demonstrate that SPIM can be applied to visualize the
embryogenesis of the relatively opaque Drosophila melanogaster
in vivo.
BibTeX:
@article{Huisken2004-vu,
  author = {Huisken, Jan and Swoger, Jim and Del Bene, Filippo and
Wittbrodt, Joachim and Stelzer, Ernst H K}, title = {Optical sectioning deep inside live embryos by selective plane
illumination microscopy}, journal = {Science}, year = {2004}, volume = {305}, number = {5686}, pages = {1007--1009}, url = {http://dx.doi.org/10.1126/science.1100035}, doi = {http://dx.doi.org/10.1126/science.1100035} }
Huotari, S., Pylkkänen, T., Verbeni, R., Monaco, G. and Hämäläinen, K. Direct tomography with chemical-bond contrast 2011 Nat. Mater.
Vol. 10(7), pp. 489-493 
article DOI URL 
Abstract: Three-dimensional (3D) X-ray imaging methods have advanced
tremendously during recent years. Traditional tomography uses
absorption as the contrast mechanism, but for many purposes
its sensitivity is limited. The introduction of diffraction,
small-angle scattering, refraction, and phase contrasts has
increased the sensitivity, especially in materials composed of
light elements (for example, carbon and oxygen). X-ray
spectroscopy, in principle, offers information on element
composition and chemical environment. However, its application
in 3D imaging over macroscopic length scales has not been
possible for light elements. Here we introduce a new
hard-X-ray spectroscopic tomography with a unique sensitivity
to light elements. In this method, dark-field section images
are obtained directly without any reconstruction algorithms.
We apply the method to acquire the 3D structure and map the
chemical bonding in selected samples relevant to materials
science. The novel aspects make this technique a powerful new
imaging tool, with an inherent access to the molecular-level
chemical environment.
BibTeX:
@article{Huotari2011-za,
  author = {Huotari, Simo and Pylkkänen, Tuomas and Verbeni, Roberto
and Monaco, Giulio and Hämäläinen, Keijo}, title = {Direct tomography with chemical-bond contrast}, journal = {Nat. Mater.}, year = {2011}, volume = {10}, number = {7}, pages = {489--493}, url = {http://dx.doi.org/10.1038/nmat3031}, doi = {http://dx.doi.org/10.1038/nmat3031} }
Ichikawa, T., Nakazato, K., Keller, P.J., Kajiura-Kobayashi, H., Stelzer, E.H.K., Mochizuki, A. and Nonaka, S. Live imaging and quantitative analysis of gastrulation in
mouse embryos using light-sheet microscopy and 3D tracking
tools
2014 Nat. Protoc.
Vol. 9(3), pp. 575-585 
article DOI URL 
Abstract: This protocol describes how to observe gastrulation in living
mouse embryos by using light-sheet microscopy and
computational tools to analyze the resulting image data at the
single-cell level. We describe a series of techniques needed
to image the embryos under physiological conditions, including
how to hold mouse embryos without agarose embedding, how to
transfer embryos without air exposure and how to construct
environmental chambers for live imaging by digital scanned
light-sheet microscopy (DSLM). Computational tools include
manual and semiautomatic tracking programs that are developed
for analyzing the large 4D data sets acquired with this
system. Note that this protocol does not include details of
how to build the light-sheet microscope itself. Time-lapse
imaging ends within 12 h, with subsequent tracking analysis
requiring 3-6 d. Other than some mouse-handling skills, this
protocol requires no advanced skills or knowledge. Light-sheet
microscopes are becoming more widely available, and thus the
techniques outlined in this paper should be helpful for
investigating mouse embryogenesis.
BibTeX:
@article{Ichikawa2014-wd,
  author = {Ichikawa, Takehiko and Nakazato, Kenichi and Keller, Philipp J
and Kajiura-Kobayashi, Hiroko and Stelzer, Ernst H K and
Mochizuki, Atsushi and Nonaka, Shigenori}, title = {Live imaging and quantitative analysis of gastrulation in
mouse embryos using light-sheet microscopy and 3D tracking
tools}, journal = {Nat. Protoc.}, year = {2014}, volume = {9}, number = {3}, pages = {575--585}, url = {http://dx.doi.org/10.1038/nprot.2014.035}, doi = {http://dx.doi.org/10.1038/nprot.2014.035} }
Ichikawa, T., Nakazato, K., Keller, P.J., Kajiura-Kobayashi, H., Stelzer, E.H.K., Mochizuki, A. and Nonaka, S. Live imaging of whole mouse embryos during gastrulation:
migration analyses of epiblast and mesodermal cells
2013 PLoS One
Vol. 8(7), pp. e64506 
article DOI URL 
Abstract: During gastrulation in the mouse embryo, dynamic cell
movements including epiblast invagination and mesodermal layer
expansion lead to the establishment of the three-layered body
plan. The precise details of these movements, however, are
sometimes elusive, because of the limitations in live imaging.
To overcome this problem, we developed techniques to enable
observation of living mouse embryos with digital scanned light
sheet microscope (DSLM). The achieved deep and high
time-resolution images of GFP-expressing nuclei and following
3D tracking analysis revealed the following findings: (i)
Interkinetic nuclear migration (INM) occurs in the epiblast at
embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in
the E5.5 embryo, when the epiblast is a monolayer and not yet
pseudostratified. (iii) Primary driving force for INM at E6.5
is not pressure from neighboring nuclei. (iv) Mesodermal cells
migrate not as a sheet but as individual cells without
coordination.
BibTeX:
@article{Ichikawa2013-tz,
  author = {Ichikawa, Takehiko and Nakazato, Kenichi and Keller, Philipp J
and Kajiura-Kobayashi, Hiroko and Stelzer, Ernst H K and
Mochizuki, Atsushi and Nonaka, Shigenori}, title = {Live imaging of whole mouse embryos during gastrulation:
migration analyses of epiblast and mesodermal cells}, journal = {PLoS One}, year = {2013}, volume = {8}, number = {7}, pages = {e64506}, url = {http://dx.doi.org/10.1371/journal.pone.0064506}, doi = {http://dx.doi.org/10.1371/journal.pone.0064506} }
Ingaramo, M., York, A.G., Hoogendoorn, E., Postma, M., Shroff, H. and Patterson, G.H. Richardson-Lucy deconvolution as a general tool for
combining images with complementary strengths
2014 Chemphyschem
Vol. 15(4), pp. 794-800 
article DOI URL 
Abstract: We use Richardson-Lucy (RL) deconvolution to combine multiple
images of a simulated object into a single image in the
context of modern fluorescence microscopy techniques. RL
deconvolution can merge images with very different
point-spread functions, such as in multiview light-sheet
microscopes,1, 2 while preserving the best resolution
information present in each image. We show that RL
deconvolution is also easily applied to merge high-resolution,
high-noise images with low-resolution, low-noise images,
relevant when complementing conventional microscopy with
localization microscopy. We also use RL deconvolution to merge
images produced by different simulated illumination patterns,
relevant to structured illumination microscopy (SIM)3, 4 and
image scanning microscopy (ISM). The quality of our ISM
reconstructions is at least as good as reconstructions using
standard inversion algorithms for ISM data, but our method
follows a simpler recipe that requires no mathematical
insight. Finally, we apply RL deconvolution to merge a series
of ten images with varying signal and resolution levels. This
combination is relevant to gated stimulated-emission depletion
(STED) microscopy, and shows that merges of high-quality
images are possible even in cases for which a non-iterative
inversion algorithm is unknown.
BibTeX:
@article{Ingaramo2014-sr,
  author = {Ingaramo, Maria and York, Andrew G and Hoogendoorn, Eelco and
Postma, Marten and Shroff, Hari and Patterson, George H}, title = {Richardson-Lucy deconvolution as a general tool for
combining images with complementary strengths}, journal = {Chemphyschem}, publisher = {WILEY-VCH Verlag}, year = {2014}, volume = {15}, number = {4}, pages = {794--800}, url = {http://dx.doi.org/10.1002/cphc.201300831}, doi = {http://dx.doi.org/10.1002/cphc.201300831} }
Jährling, N., Becker, K. and Dodt, H.-U. 3D-reconstruction of blood vessels by ultramicroscopy 2009 Organogenesis
Vol. 5(4), pp. 227-230 
article URL 
Abstract: As recently shown, ultramicroscopy (UM) allows
3D-visualization of even large microscopic structures with
microm resolution. Thus, it can be applied to anatomical
studies of numerous biological and medical specimens. We
reconstructed the three-dimensional architecture of
tomato-lectin (Lycopersicon esculentum) stained vascular
networks by UM in whole mouse organs. The topology of filigree
branches of the microvasculature was visualized. Since tumors
require an extensive growth of blood vessels to survive, this
novel approach may open up new vistas in neurobiology and
histology, particularly in cancer research.
BibTeX:
@article{Jahrling2009-pm,
  author = {Jährling, Nina and Becker, Klaus and Dodt, Hans-Ulrich},
  title = {3D-reconstruction of blood vessels by ultramicroscopy},
  journal = {Organogenesis},
  year = {2009},
  volume = {5},
  number = {4},
  pages = {227--230},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/20539742}
}
Jährling, N., Becker, K., Schönbauer, C., Schnorrer, F. and Dodt, H.-U. Three-dimensional reconstruction and segmentation of intact
Drosophila by ultramicroscopy
2010 Front. Syst. Neurosci.
Vol. 4, pp. 1 
article DOI URL 
Abstract: Genetic mutants are invaluable for understanding the
development, physiology and behaviour of Drosophila. Modern
molecular genetic techniques enable the rapid generation of
large numbers of different mutants. To phenotype these mutants
sophisticated microscopy techniques are required, ideally
allowing the 3D-reconstruction of the anatomy of an adult fly
from a single scan. Ultramicroscopy enables up to cm fields of
view, whilst providing micron resolution. In this paper, we
present ultramicroscopy reconstructions of the flight
musculature, the nervous system, and the digestive tract of
entire, chemically cleared, drosophila in autofluorescent
light. The 3D-reconstructions thus obtained verify that the
anatomy of a whole fly, including the filigree spatial
organization of the direct flight muscles, can be analysed
from a single ultramicroscopy reconstruction. The recording
procedure, including 3D-reconstruction using standard
software, takes no longer than 30 min. Additionally, image
segmentation, which would allow for further quantitative
analysis, was performed.
BibTeX:
@article{Jahrling2010-sx,
  author = {Jährling, Nina and Becker, Klaus and Schönbauer,
Cornelia and Schnorrer, Frank and Dodt, Hans-Ulrich}, title = {Three-dimensional reconstruction and segmentation of intact
Drosophila by ultramicroscopy}, journal = {Front. Syst. Neurosci.}, year = {2010}, volume = {4}, pages = {1}, url = {http://dx.doi.org/10.3389/neuro.06.001.2010}, doi = {http://dx.doi.org/10.3389/neuro.06.001.2010} }
Jahr, W., Schmid, B., Schmied, C., Fahrbach, F.O. and Huisken, J. Hyperspectral light sheet microscopy 2015 Nat. Commun.
Vol. 6, pp. 7990 
article DOI URL 
Abstract: To study the development and interactions of cells and
tissues, multiple fluorescent markers need to be imaged
efficiently in a single living organism. Instead of acquiring
individual colours sequentially with filters, we created a
platform based on line-scanning light sheet microscopy to
record the entire spectrum for each pixel in a
three-dimensional volume. We evaluated data sets with varying
spectral sampling and determined the optimal channel width to
be around 5 nm. With the help of these data sets, we show that
our setup outperforms filter-based approaches with regard to
image quality and discrimination of fluorophores. By spectral
unmixing we resolved overlapping fluorophores with up to
nanometre resolution and removed autofluorescence in zebrafish
and fruit fly embryos.
BibTeX:
@article{Jahr2015-th,
  author = {Jahr, Wiebke and Schmid, Benjamin and Schmied, Christopher and
Fahrbach, Florian O and Huisken, Jan}, title = {Hyperspectral light sheet microscopy}, journal = {Nat. Commun.}, publisher = {Nature Publishing Group}, year = {2015}, volume = {6}, pages = {7990}, url = {http://dx.doi.org/10.1038/ncomms8990}, doi = {http://dx.doi.org/10.1038/ncomms8990} }
Jeandupeux, E., Lobjois, V. and Ducommun, B. 3D print customized sample holders for live light sheet
microscopy
2015 Biochem. Biophys. Res. Commun.
Vol. 463(4), pp. 1141-1143 
article DOI URL 
Abstract: A major hurdle to the widespread application of light sheet
microscopy is the lack of versatile and non-intrusive sample
holders that are adaptable to a variety of biological samples
for live imaging. To overcome this limitation, we present
herein the application of 3D printing to the fabrication of a
fully customizable casting kit. 3D printing enables facile
preparation of hydrogel sample holders adaptable to any shape
and number of specimen. As an example, we present the use of
this device to produce a four-sample holder adapted to
parallel live monitoring of multicellular tumor spheroid
growth. To share our solution with the light sheet microscopy
community, all files necessary to produce or customize sample
holders are freely available online.
BibTeX:
@article{Jeandupeux2015-yc,
  author = {Jeandupeux, Emeric and Lobjois, Valérie and Ducommun,
Bernard}, title = {3D print customized sample holders for live light sheet
microscopy}, journal = {Biochem. Biophys. Res. Commun.}, year = {2015}, volume = {463}, number = {4}, pages = {1141--1143}, url = {http://dx.doi.org/10.1016/j.bbrc.2015.06.072}, doi = {http://dx.doi.org/10.1016/j.bbrc.2015.06.072} }
Jemielita, M., Taormina, M.J., Burns, A.R., Hampton, J.S., Rolig, A.S., Guillemin, K. and Parthasarathy, R. Spatial and temporal features of the growth of a bacterial
species colonizing the zebrafish gut
2014 MBio
Vol. 5(6) 
article DOI URL 
Abstract: UNLABELLED: The vertebrate intestine is home to microbial
ecosystems that play key roles in host development and health.
Little is known about the spatial and temporal dynamics of
these microbial communities, limiting our understanding of
fundamental properties, such as their mechanisms of growth,
propagation, and persistence. To address this, we inoculated
initially germ-free zebrafish larvae with fluorescently
labeled strains of an Aeromonas species, representing an
abundant genus in the zebrafish gut. Using light sheet
fluorescence microscopy to obtain three-dimensional images
spanning the gut, we quantified the entire bacterial load, as
founding populations grew from tens to tens of thousands of
cells over several hours. The data yield the first ever
measurements of the growth kinetics of a microbial species
inside a live vertebrate intestine and show dynamics that
robustly fit a logistic growth model. Intriguingly, bacteria
were nonuniformly distributed throughout the gut, and
bacterial aggregates showed considerably higher growth rates
than did discrete individuals. The form of aggregate growth
indicates intrinsically higher division rates for clustered
bacteria, rather than surface-mediated agglomeration onto
clusters. Thus, the spatial organization of gut bacteria both
relative to the host and to each other impacts overall growth
kinetics, suggesting that spatial characterizations will be an
important input to predictive models of host-associated
microbial community assembly. IMPORTANCE: Our intestines are
home to vast numbers of microbes that influence many aspects
of health and disease. Though we now know a great deal about
the constituents of the gut microbiota, we understand very
little about their spatial structure and temporal dynamics in
humans or in any animal: how microbial populations establish
themselves, grow, fluctuate, and persist. To address this, we
made use of a model organism, the zebrafish, and a new optical
imaging technique, light sheet fluorescence microscopy, to
visualize for the first time the colonization of a live,
vertebrate gut by specific bacteria with sufficient resolution
to quantify the population over a range from a few individuals
to tens of thousands of bacterial cells. Our results provide
unprecedented measures of bacterial growth kinetics and also
show the influence of spatial structure on bacterial
populations, which can be revealed only by direct imaging.
BibTeX:
@article{Jemielita2014-ut,
  author = {Jemielita, Matthew and Taormina, Michael J and Burns, Adam R
and Hampton, Jennifer S and Rolig, Annah S and Guillemin,
Karen and Parthasarathy, Raghuveer}, title = {Spatial and temporal features of the growth of a bacterial
species colonizing the zebrafish gut}, journal = {MBio}, year = {2014}, volume = {5}, number = {6}, url = {http://dx.doi.org/10.1128/mBio.01751-14}, doi = {http://dx.doi.org/10.1128/mBio.01751-14} }
Jemielita, M., Taormina, M.J., Delaurier, A., Kimmel, C.B. and Parthasarathy, R. Comparing phototoxicity during the development of a zebrafish
craniofacial bone using confocal and light sheet fluorescence
microscopy techniques
2013 J. Biophotonics
Vol. 6(11-12), pp. 920-928 
article DOI URL 
Abstract: The combination of genetically encoded fluorescent proteins
and three-dimensional imaging enables cell-type-specific
studies of embryogenesis. Light sheet microscopy, in which
fluorescence excitation is provided by a plane of laser light,
is an appealing approach to live imaging due to its high speed
and efficient use of photons. While the advantages of rapid
imaging are apparent from recent work, the importance of low
light levels to studies of development is not well
established. We examine the zebrafish opercle, a craniofacial
bone that exhibits pronounced shape changes at early
developmental stages, using both spinning disk confocal and
light sheet microscopies of fluorescent osteoblast cells. We
find normal and aberrant opercle morphologies for specimens
imaged with short time intervals using light sheet and
spinning disk confocal microscopies, respectively, under
equivalent exposure conditions over developmentally-relevant
time scales. Quantification of shapes reveals that the
differently imaged specimens travel along distinct
trajectories in morphological space.
BibTeX:
@article{Jemielita2013-is,
  author = {Jemielita, Matthew and Taormina, Michael J and Delaurier,
April and Kimmel, Charles B and Parthasarathy, Raghuveer}, title = {Comparing phototoxicity during the development of a zebrafish
craniofacial bone using confocal and light sheet fluorescence
microscopy techniques}, journal = {J. Biophotonics}, year = {2013}, volume = {6}, number = {11-12}, pages = {920--928}, url = {http://dx.doi.org/10.1002/jbio.201200144}, doi = {http://dx.doi.org/10.1002/jbio.201200144} }
Jorand, R., Le Corre, G., Andilla, J., Maandhui, A., Frongia, C., Lobjois, V., Ducommun, B. and Lorenzo, C. Deep and clear optical imaging of thick inhomogeneous samples 2012 PLoS One
Vol. 7(4), pp. e35795 
article DOI URL 
Abstract: Inhomogeneity in thick biological specimens results in poor
imaging by light microscopy, which deteriorates as the focal
plane moves deeper into the specimen. Here, we have combined
selective plane illumination microscopy (SPIM) with wavefront
sensor adaptive optics (wao). Our waoSPIM is based on a direct
wavefront measure using a Hartmann-Shack wavefront sensor and
fluorescent beads as point source emitters. We demonstrate the
use of this waoSPIM method to correct distortions in
three-dimensional biological imaging and to improve the
quality of images from deep within thick inhomogeneous
samples.
BibTeX:
@article{Jorand2012-jx,
  author = {Jorand, Raphael and Le Corre, Gwénaële and Andilla,
Jordi and Maandhui, Amina and Frongia, Céline and Lobjois,
Valérie and Ducommun, Bernard and Lorenzo, Corinne}, title = {Deep and clear optical imaging of thick inhomogeneous samples}, journal = {PLoS One}, year = {2012}, volume = {7}, number = {4}, pages = {e35795}, url = {http://dx.doi.org/10.1371/journal.pone.0035795}, doi = {http://dx.doi.org/10.1371/journal.pone.0035795} }
Junker, J.P., Noël, E.S., Guryev, V., Peterson, K.A., Shah, G., Huisken, J., McMahon, A.P., Berezikov, E., Bakkers, J. and van Oudenaarden, A. Genome-wide RNA Tomography in the zebrafish embryo 2014 Cell
Vol. 159(3), pp. 662-675 
article DOI URL 
Abstract: Advancing our understanding of embryonic development is
heavily dependent on identification of novel pathways or
regulators. Although genome-wide techniques such as RNA
sequencing are ideally suited for discovering novel candidate
genes, they are unable to yield spatially resolved information
in embryos or tissues. Microscopy-based approaches, using in
situ hybridization, for example, can provide spatial
information about gene expression, but are limited to
analyzing one or a few genes at a time. Here, we present a
method where we combine traditional histological techniques
with low-input RNA sequencing and mathematical image
reconstruction to generate a high-resolution genome-wide 3D
atlas of gene expression in the zebrafish embryo at three
developmental stages. Importantly, our technique enables
searching for genes that are expressed in specific spatial
patterns without manual image annotation. We envision broad
applicability of RNA tomography as an accurate and sensitive
approach for spatially resolved transcriptomics in whole
embryos and dissected organs.
BibTeX:
@article{Junker2014-qg,
  author = {Junker, Jan Philipp and Noël, Emily S and Guryev, Victor
and Peterson, Kevin A and Shah, Gopi and Huisken, Jan and
McMahon, Andrew P and Berezikov, Eugene and Bakkers, Jeroen
and van Oudenaarden, Alexander}, title = {Genome-wide RNA Tomography in the zebrafish embryo}, journal = {Cell}, year = {2014}, volume = {159}, number = {3}, pages = {662--675}, url = {http://dx.doi.org/10.1016/j.cell.2014.09.038}, doi = {http://dx.doi.org/10.1016/j.cell.2014.09.038} }
Köster, I. and Haas, P. Light Sheet Microscopy Turned Vertically 2015 Optik & Photonik
Vol. 10(4), pp. 39-43 
article DOI URL 
Abstract: Living cells and organisms often suffer from the high light
intensities used for fluorescent imaging. Light sheet microscopy
reduces phototoxic effects and bleaching by illuminating a
specimen in only a single plane at a time. A new light sheet
microscope combines light sheet and confocal microscopy in one
system without compromising either functionality and allows the
combination of the two methods, e.g. confocal photomanipulation
with subsequent light sheet acquisition, for new applications.
BibTeX:
@article{Koster2015-sc,
  author = {Köster, Isabelle and Haas, Petra},
  title = {Light Sheet Microscopy Turned Vertically},
  journal = {Optik & Photonik},
  publisher = {WILEY-VCH Verlag},
  year = {2015},
  volume = {10},
  number = {4},
  pages = {39--43},
  url = {http://dx.doi.org/10.1002/opph.201500028},
  doi = {http://dx.doi.org/10.1002/opph.201500028}
}
Kalchmair, S., Jährling, N., Becker, K. and Dodt, H.-U. Image contrast enhancement in confocal ultramicroscopy 2010 Opt. Lett.
Vol. 35(1), pp. 79-81 
article DOI URL 
Abstract: Ultramicroscopy allows for the 3D reconstruction of centimeter
sized samples with a spatial resolution of several
micrometers. Nevertheless, in poorly cleared or very large
specimens the images may suffer from blurring and low contrast
levels. To address these problems, ultramicroscopy was
combined with the principle of confocal microscopy using a
slowly rotating Nipkow disk. This configuration was tested by
comparing images from mouse hippocampal neurons and mouse
liver blood vessels recorded in confocal and conventional
mode. It was found that confocality minimizes the background
noise and considerably improves the signal-to-noise ratio when
applied to ultramicroscopy.
BibTeX:
@article{Kalchmair2010-lb,
  author = {Kalchmair, Stefan and Jährling, Nina and Becker, Klaus
and Dodt, Hans-Ulrich}, title = {Image contrast enhancement in confocal ultramicroscopy}, journal = {Opt. Lett.}, year = {2010}, volume = {35}, number = {1}, pages = {79--81}, url = {http://dx.doi.org/10.1364/OL.35.000079}, doi = {http://dx.doi.org/10.1364/OL.35.000079} }
Karaköylü, E.M., Franks, P.J.S., Tanaka, Y., Roberts, P.L.D. and Jaffe, J.S. Copepod feeding quantified by planar laser imaging of gut
fluorescence
2009 Limnol. Oceanogr. Methods
Vol. 7(1), pp. 33-41 
article DOI URL 
Abstract: We present a new method for quantifying the feeding of individual
copepods, using a planar sheet of laser light to stimulate the
fluorescence of phytoplankton ingested by the copepod. The
fluorescence is imaged with a sensitive CCD camera, giving
two-dimensional images of the copepod's gut with 20
20 µm spatial resolution. Using tethered copepods, we have
obtained > 3 h long time series of copepod gut fluorescence with
images every 15--20 s. The same individual copepod can be used
for multiple experiments, obviating the problems of individual
variability as a source of error. Initial data reveal two
distinct patterns of variability as material moves through two
functionally different gut compartments. These patterns reflect
processes occurring in each compartment. The upper (anterior)
mid-gut shows higher variability and less repeatability than the
posterior midgut where undigested material is aggregated into a
fecal pellet and evacuated at regular intervals. Variability in
the upper mid-gut is likely due to factors such as intermittence
of feeding and relatively complex mixing dynamics. In the
posterior mid-gut, mixing dynamics are much simpler, and the
variability of the upper compartment is integrated over the time
scale of pellet formation.
BibTeX:
@article{Karakoylu2009-rz,
  author = {Karaköylü, Erdem M and Franks, Peter J S and Tanaka,
Yuji and Roberts, Paul L D and Jaffe, Jules S}, title = {Copepod feeding quantified by planar laser imaging of gut
fluorescence}, journal = {Limnol. Oceanogr. Methods}, year = {2009}, volume = {7}, number = {1}, pages = {33--41}, url = {http://dx.doi.org/10.4319/lom.2009.7.33}, doi = {http://dx.doi.org/10.4319/lom.2009.7.33} }
Kaufmann, A., Mickoleit, M., Weber, M. and Huisken, J. Multilayer mounting enables long-term imaging of zebrafish
development in a light sheet microscope
2012 Development
Vol. 139(17), pp. 3242-3247 
article DOI URL 
Abstract: Light sheet microscopy techniques, such as selective plane
illumination microscopy (SPIM), are ideally suited for
time-lapse imaging of developmental processes lasting several
hours to a few days. The success of this promising technology
has mainly been limited by the lack of suitable techniques for
mounting fragile samples. Embedding zebrafish embryos in
agarose, which is common in conventional confocal microscopy,
has resulted in severe growth defects and unreliable results.
In this study, we systematically quantified the viability and
mobility of zebrafish embryos mounted under more suitable
conditions. We found that tubes made of fluorinated ethylene
propylene (FEP) filled with low concentrations of agarose or
methylcellulose provided an optimal balance between sufficient
confinement of the living embryo in a physiological
environment over 3 days and optical clarity suitable for
fluorescence imaging. We also compared the effect of different
concentrations of Tricaine on the development of zebrafish and
provide guidelines for its optimal use depending on the
application. Our results will make light sheet microscopy
techniques applicable to more fields of developmental biology,
in particular the multiview long-term imaging of zebrafish
embryos and other small organisms. Furthermore, the refinement
of sample preparation for in toto and in vivo imaging will
promote other emerging optical imaging techniques, such as
optical projection tomography (OPT).
BibTeX:
@article{Kaufmann2012-xp,
  author = {Kaufmann, Anna and Mickoleit, Michaela and Weber, Michael and
Huisken, Jan}, title = {Multilayer mounting enables long-term imaging of zebrafish
development in a light sheet microscope}, journal = {Development}, year = {2012}, volume = {139}, number = {17}, pages = {3242--3247}, url = {http://dx.doi.org/10.1242/dev.082586}, doi = {http://dx.doi.org/10.1242/dev.082586} }
Keller, P.J. In vivo imaging of zebrafish embryogenesis 2013 Methods
Vol. 62(3), pp. 268-278 
article DOI URL 
Abstract: The zebrafish Danio rerio has emerged as a powerful vertebrate
model system that lends itself particularly well to
quantitative investigations with live imaging approaches,
owing to its exceptionally high optical clarity in embryonic
and larval stages. Recent advances in light microscopy
technology enable comprehensive analyses of cellular dynamics
during zebrafish embryonic development, systematic mapping of
gene expression dynamics, quantitative reconstruction of
mutant phenotypes and the system-level biophysical study of
morphogenesis. Despite these technical breakthroughs, it
remains challenging to design and implement experiments for in
vivo long-term imaging at high spatio-temporal resolution.
This article discusses the fundamental challenges in zebrafish
long-term live imaging, provides experimental protocols and
highlights key properties and capabilities of advanced
fluorescence microscopes. The article focuses in particular on
experimental assays based on light sheet-based fluorescence
microscopy, an emerging imaging technology that achieves
exceptionally high imaging speeds and excellent
signal-to-noise ratios, while minimizing light-induced damage
to the specimen. This unique combination of capabilities makes
light sheet microscopy an indispensable tool for the in vivo
long-term imaging of large developing organisms.
BibTeX:
@article{Keller2013-zv,
  author = {Keller, Philipp J},
  title = {In vivo imaging of zebrafish embryogenesis},
  journal = {Methods},
  year = {2013},
  volume = {62},
  number = {3},
  pages = {268--278},
  url = {http://dx.doi.org/10.1016/j.ymeth.2013.03.015},
  doi = {http://dx.doi.org/10.1016/j.ymeth.2013.03.015}
}
Keller, P.J. and Ahrens, M.B. Visualizing whole-brain activity and development at the
single-cell level using light-sheet microscopy
2015 Neuron
Vol. 85(3), pp. 462-483 
article DOI URL 
Abstract: The nature of nervous system function and development is
inherently global, since all components eventually influence
one another. Networks communicate through dense synaptic,
electric, and modulatory connections and develop through
concurrent growth and interlinking of their neurons,
processes, glia, and blood vessels. These factors drive the
development of techniques capable of imaging neural signaling,
anatomy, and developmental processes at ever-larger scales.
Here, we discuss the nature of questions benefitting from
large-scale imaging techniques and introduce recent
applications. We focus on emerging light-sheet microscopy
approaches, which are well suited for live imaging of large
systems with high spatiotemporal resolution and over long
periods of time. We also discuss computational methods
suitable for extracting biological information from the
resulting system-level image data sets. Together with new
tools for reporting and manipulating neuronal activity and
gene expression, these techniques promise new insights into
the large-scale function and development of neural systems.
BibTeX:
@article{Keller2015-ge,
  author = {Keller, Philipp J and Ahrens, Misha B},
  title = {Visualizing whole-brain activity and development at the
single-cell level using light-sheet microscopy}, journal = {Neuron}, year = {2015}, volume = {85}, number = {3}, pages = {462--483}, url = {http://dx.doi.org/10.1016/j.neuron.2014.12.039}, doi = {http://dx.doi.org/10.1016/j.neuron.2014.12.039} }
Keller, P.J., Ahrens, M.B. and Freeman, J. Light-sheet imaging for systems neuroscience 2015 Nat. Methods
Vol. 12(1), pp. 27-29 
article DOI URL 
BibTeX:
@article{Keller2015-md,
  author = {Keller, Philipp J and Ahrens, Misha B and Freeman, Jeremy},
  title = {Light-sheet imaging for systems neuroscience},
  journal = {Nat. Methods},
  year = {2015},
  volume = {12},
  number = {1},
  pages = {27--29},
  url = {http://dx.doi.org/10.1038/nmeth.3214},
  doi = {http://dx.doi.org/10.1038/nmeth.3214}
}
Keller, P.J. and Dodt, H.-U. Light sheet microscopy of living or cleared specimens 2012 Curr. Opin. Neurobiol.
Vol. 22(1), pp. 138-143 
article DOI URL 
Abstract: Light sheet microscopy is a versatile imaging technique with a
unique combination of capabilities. It provides high imaging
speed, high signal-to-noise ratio and low levels of
photobleaching and phototoxic effects. These properties are
crucial in a wide range of applications in the life sciences,
from live imaging of fast dynamic processes in single cells to
long-term observation of developmental dynamics in entire
large organisms. When combined with tissue clearing methods,
light sheet microscopy furthermore allows rapid imaging of
large specimens with excellent coverage and high spatial
resolution. Even samples up to the size of entire mammalian
brains can be efficiently recorded and quantitatively
analyzed. Here, we provide an overview of the history of light
sheet microscopy, review the development of tissue clearing
methods, and discuss recent technical breakthroughs that have
the potential to influence the future direction of the field.
BibTeX:
@article{Keller2012-xl,
  author = {Keller, Philipp J and Dodt, Hans-Ulrich},
  title = {Light sheet microscopy of living or cleared specimens},
  journal = {Curr. Opin. Neurobiol.},
  publisher = {Elsevier Ltd},
  year = {2012},
  volume = {22},
  number = {1},
  pages = {138--143},
  url = {http://dx.doi.org/10.1016/j.conb.2011.08.003},
  doi = {http://dx.doi.org/10.1016/j.conb.2011.08.003}
}
Keller, P.J., Pampaloni, F., Lattanzi, G. and Stelzer, E.H.K. Three-dimensional microtubule behavior in Xenopus egg extracts
reveals four dynamic states and state-dependent elastic
properties
2008 Biophys. J.
Vol. 95(3), pp. 1474-1486 
article DOI URL 
Abstract: Although microtubules are key players in many cellular
processes, very little is known about their dynamic and
mechanical properties in physiological three-dimensional
environments. The conventional model of microtubule dynamic
instability postulates two dynamic microtubule states, growth
and shrinkage. However, several studies have indicated that
such a model does not provide a comprehensive quantitative and
qualitative description of microtubule behavior. Using
three-dimensional laser light-sheet fluorescence microscopy
and a three-dimensional sample preparation in spacious Teflon
cylinders, we measured microtubule dynamic instability and
elasticity in interphase Xenopus laevis egg extracts. Our data
are inconsistent with a two-state model of microtubule dynamic
instability and favor an extended four-state model with two
independent metastable pause states over a three-state model
with a single pause state. Moreover, our data on kinetic state
transitions rule out a simple GTP cap model as the driving
force of microtubule stabilization in egg extracts on
timescales of a few seconds or longer. We determined the
three-dimensional elastic properties of microtubules as a
function of both the contour length and the dynamic state. Our
results indicate that pausing microtubules are less flexible
than growing microtubules and suggest a growth-speed-dependent
persistence length. These data might hint toward mechanisms
that enable microtubules to efficiently perform multiple
different tasks in the cell and suggest the development of a
unified model of microtubule dynamics and microtubule
mechanics.
BibTeX:
@article{Keller2008-bp,
  author = {Keller, Philipp J and Pampaloni, Francesco and Lattanzi,
Gianluca and Stelzer, Ernst H K}, title = {Three-dimensional microtubule behavior in Xenopus egg extracts
reveals four dynamic states and state-dependent elastic
properties}, journal = {Biophys. J.}, year = {2008}, volume = {95}, number = {3}, pages = {1474--1486}, url = {http://dx.doi.org/10.1529/biophysj.107.128223}, doi = {http://dx.doi.org/10.1529/biophysj.107.128223} }
Keller, P.J., Pampaloni, F. and Stelzer, E.H. Life sciences require the third dimension 2006 Curr. Opin. Cell Biol.
Vol. 18(1), pp. 117-124 
article DOI URL 
Abstract: Novel technologies are required for three-dimensional cell
biology and biophysics. By three-dimensional we refer to
experimental conditions that essentially try to avoid hard and
flat surfaces and favour unconstrained sample dynamics. We
believe that light-sheet-based microscopes are particularly
well suited to studies of sensitive three-dimensional
biological systems. The application of such instruments can be
illustrated with examples from the biophysics of microtubule
dynamics and three-dimensional cell cultures. Our experience
leads us to suggest that three-dimensional approaches reveal
new aspects of a system and enable experiments to be performed
in a more physiological and hence clinically more relevant
context.
BibTeX:
@article{Keller2006-cx,
  author = {Keller, Philipp J and Pampaloni, Francesco and Stelzer, Ernst
Hk}, title = {Life sciences require the third dimension}, journal = {Curr. Opin. Cell Biol.}, year = {2006}, volume = {18}, number = {1}, pages = {117--124}, url = {http://dx.doi.org/10.1016/j.ceb.2005.12.012}, doi = {http://dx.doi.org/10.1016/j.ceb.2005.12.012} }
Keller, P.J., Pampaloni, F. and Stelzer, E.H.K. Three-dimensional preparation and imaging reveal intrinsic
microtubule properties
2007 Nat. Methods
Vol. 4(10), pp. 843-846 
article DOI URL 
Abstract: We present an experimental investigation of microtubule
dynamic instability in three dimensions, based on laser
light-sheet fluorescence microscopy. We introduce
three-dimensional (3D) preparation of Xenopus laevis egg
extracts in Teflon-based cylinders and provide algorithms for
3D image processing. Our approach gives experimental access to
the intrinsic dynamic properties of microtubules and to
microtubule population statistics in single asters. We obtain
evidence for a stochastic nature of microtubule pausing.
BibTeX:
@article{Keller2007-ri,
  author = {Keller, Philipp J and Pampaloni, Francesco and Stelzer, Ernst
H K}, title = {Three-dimensional preparation and imaging reveal intrinsic
microtubule properties}, journal = {Nat. Methods}, year = {2007}, volume = {4}, number = {10}, pages = {843--846}, url = {http://dx.doi.org/10.1038/nmeth1087}, doi = {http://dx.doi.org/10.1038/nmeth1087} }
Keller, P.J., Schmidt, A.D., Santella, A., Khairy, K., Bao, Z., Wittbrodt, J. and Stelzer, E.H.K. Fast, high-contrast imaging of animal development with scanned
light sheet-based structured-illumination microscopy
2010 Nat. Methods
Vol. 7(8), pp. 637-642 
article DOI URL 
Abstract: Recording light-microscopy images of large, nontransparent
specimens, such as developing multicellular organisms, is
complicated by decreased contrast resulting from light
scattering. Early zebrafish development can be captured by
standard light-sheet microscopy, but new imaging strategies
are required to obtain high-quality data of late development
or of less transparent organisms. We combined digital scanned
laser light-sheet fluorescence microscopy with incoherent
structured-illumination microscopy (DSLM-SI) and created
structured-illumination patterns with continuously adjustable
frequencies. Our method discriminates the specimen-related
scattered background from signal fluorescence, thereby
removing out-of-focus light and optimizing the contrast of
in-focus structures. DSLM-SI provides rapid control of the
illumination pattern, exceptional imaging quality and high
imaging speeds. We performed long-term imaging of zebrafish
development for 58 h and fast multiple-view imaging of early
Drosophila melanogaster development. We reconstructed cell
positions over time from the Drosophila DSLM-SI data and
created a fly digital embryo.
BibTeX:
@article{Keller2010-jh,
  author = {Keller, Philipp J and Schmidt, Annette D and Santella, Anthony
and Khairy, Khaled and Bao, Zhirong and Wittbrodt, Joachim and
Stelzer, Ernst H K}, title = {Fast, high-contrast imaging of animal development with scanned
light sheet-based structured-illumination microscopy}, journal = {Nat. Methods}, year = {2010}, volume = {7}, number = {8}, pages = {637--642}, url = {http://dx.doi.org/10.1038/nmeth.1476}, doi = {http://dx.doi.org/10.1038/nmeth.1476} }
Keller, P.J., Schmidt, A.D., Wittbrodt, J. and Stelzer, E.H.K. Digital scanned laser light-sheet fluorescence microscopy
(DSLM) of zebrafish and Drosophila embryonic development
2011 Cold Spring Harb. Protoc.
Vol. 2011(10), pp. 1235-1243 
article DOI URL 
Abstract: Embryonic development is one of the most complex processes
encountered in biology. In vertebrates and higher invertebrates,
a single cell transforms into a fully functional organism
comprising several tens of thousands of cells, arranged in
tissues and organs that perform impressive tasks. In vivo
observation of this biological process at high spatiotemporal
resolution and over long periods of time is crucial for
quantitative developmental biology. Importantly, such recordings
must be realized without compromising the physiological
development of the specimen. In digital scanned laser light-sheet
fluorescence microscopy (DSLM), a specimen is rapidly scanned
with a thin sheet of light while fluorescence is recorded
perpendicular to the axis of illumination with a camera.
Combining light-sheet technology and fast laser scanning, DSLM
delivers quantitative data for entire embryos at high
spatiotemporal resolution. Compared with confocal and two-photon
fluorescence microscopy, DSLM exposes the embryo to at least
three orders of magnitude less light energy, but still provides
up to 50 times faster imaging speeds and a 10-100-fold higher
signal-to-noise ratio. By using automated image processing
algorithms, DSLM images of embryogenesis can be converted into a
digital representation. These digital embryos permit following
cells as a function of time, revealing cell fate as well as cell
origin. By means of such analyses, developmental building plans
of tissues and organs can be determined in a whole-embryo
context. This article presents a sample preparation and imaging
protocol for studying the development of whole zebrafish and
Drosophila embryos using DSLM.
BibTeX:
@article{Keller2011-py,
  author = {Keller, Philipp J and Schmidt, Annette D and Wittbrodt, Jochen
and Stelzer, Ernst H K}, title = {Digital scanned laser light-sheet fluorescence microscopy
(DSLM) of zebrafish and Drosophila embryonic development}, journal = {Cold Spring Harb. Protoc.}, year = {2011}, volume = {2011}, number = {10}, pages = {1235--1243}, url = {http://dx.doi.org/10.1101/pdb.prot065839}, doi = {http://dx.doi.org/10.1101/pdb.prot065839} }
Keller, P.J., Schmidt, A.D., Wittbrodt, J. and Stelzer, E.H.K. The zebrafish digital embryo: in toto reconstruction of
zebrafish early embryonic development with digital scanned laser
light sheet fluorescence microscopy
2009
Vol. 7367European Conferences on Biomedical Optics, pp. 73670G-73670G-10 
inproceedings DOI URL 
BibTeX:
@inproceedings{Keller2009-bq,
  author = {Keller, Philipp J and Schmidt, Annette D and Wittbrodt, Joachim
and Stelzer, Ernst H K}, title = {The zebrafish digital embryo: in toto reconstruction of
zebrafish early embryonic development with digital scanned laser
light sheet fluorescence microscopy}, booktitle = {European Conferences on Biomedical Optics}, publisher = {International Society for Optics and Photonics}, year = {2009}, volume = {7367}, pages = {73670G--73670G--10}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=780802}, doi = {http://dx.doi.org/10.1117/12.831496} }
Keller, P.J., Schmidt, A.D., Wittbrodt, J. and Stelzer, E.H.K. Reconstruction of zebrafish early embryonic development by
scanned light sheet microscopy
2008 Science
Vol. 322(5904), pp. 1065-1069 
article DOI URL 
Abstract: A long-standing goal of biology is to map the behavior of all
cells during vertebrate embryogenesis. We developed digital
scanned laser light sheet fluorescence microscopy and recorded
nuclei localization and movement in entire wild-type and
mutant zebrafish embryos over the first 24 hours of
development. Multiview in vivo imaging at 1.5 billion voxels
per minute provides ``digital embryos,'' that is,
comprehensive databases of cell positions, divisions, and
migratory tracks. Our analysis of global cell division
patterns reveals a maternally defined initial morphodynamic
symmetry break, which identifies the embryonic body axis. We
further derive a model of germ layer formation and show that
the mesendoderm forms from one-third of the embryo's cells in
a single event. Our digital embryos, with 55 million nucleus
entries, are provided as a resource.
BibTeX:
@article{Keller2008-xk,
  author = {Keller, Philipp J and Schmidt, Annette D and Wittbrodt,
Joachim and Stelzer, Ernst H K}, title = {Reconstruction of zebrafish early embryonic development by
scanned light sheet microscopy}, journal = {Science}, year = {2008}, volume = {322}, number = {5904}, pages = {1065--1069}, url = {http://dx.doi.org/10.1126/science.1162493}, doi = {http://dx.doi.org/10.1126/science.1162493} }
Keller, P.J. and Stelzer, E.H.K. Digital scanned laser light sheet fluorescence microscopy 2010 Cold Spring Harb. Protoc.
Vol. 2010(5), pp. db.top78 
article DOI URL 
Abstract: Modern applications in the life sciences are frequently based on
in vivo imaging of biological specimens, a domain for which light
microscopy approaches are typically best suited. Often,
quantitative information must be obtained from large
multicellular organisms on the cellular or even subcellular level
and with a good temporal resolution. However, this usually
requires a combination of conflicting features: high imaging
speed, low photobleaching, and low phototoxicity in the specimen,
good three-dimensional (3D) resolution, an excellent
signal-to-noise ratio, and multiple-view imaging capability. The
latter feature refers to the capability of recording a specimen
along multiple directions, which is crucial for the imaging of
large specimens with strong light-scattering or light-absorbing
tissue properties. An imaging technique that fulfills these
requirements is essential for many key applications: For example,
studying fast cellular processes over long periods of time,
imaging entire embryos throughout development, or reconstructing
the formation of morphological defects in mutants. Here, we
discuss digital scanned laser light sheet fluorescence microscopy
(DSLM) as a novel tool for quantitative in vivo imaging in the
post-genomic era and show how this emerging technique relates to
the currently most widely applied 3D microscopy techniques in
biology: confocal fluorescence microscopy and two-photon
microscopy.
BibTeX:
@article{Keller2010-gh,
  author = {Keller, Philipp J and Stelzer, Ernst H K},
  title = {Digital scanned laser light sheet fluorescence microscopy},
  journal = {Cold Spring Harb. Protoc.},
  year = {2010},
  volume = {2010},
  number = {5},
  pages = {db.top78},
  url = {http://dx.doi.org/10.1101/pdb.top78},
  doi = {http://dx.doi.org/10.1101/pdb.top78}
}
Keller, P.J. and Stelzer, E.H.K. Quantitative in vivo imaging of entire embryos with Digital
Scanned Laser Light Sheet Fluorescence Microscopy
2008 Curr. Opin. Neurobiol.
Vol. 18(6), pp. 624-632 
article DOI URL 
Abstract: The observation of biological processes in their natural in
vivo context is a key requirement for quantitative
experimental studies in the life sciences. In many instances,
it will be crucial to achieve high temporal and spatial
resolution over long periods of time without compromising the
physiological development of the specimen. Here, we discuss
the principles underlying light sheet-based fluorescence
microscopes. The most recent implementation DSLM is a tool
optimized to deliver quantitative data for entire embryos at
high spatio-temporal resolution. We compare DSLM to the two
established light microscopy techniques: confocal and
two-photon fluorescence microscopy. DSLM provides up to 50
times higher imaging speeds and a 10-100 times higher
signal-to-noise ratio, while exposing the specimens to at
least three orders of magnitude less light energy than
confocal and two-photon fluorescence microscopes. We conclude
with a perspective for future development.
BibTeX:
@article{Keller2008-hj,
  author = {Keller, Philipp J and Stelzer, Ernst H K},
  title = {Quantitative in vivo imaging of entire embryos with Digital
Scanned Laser Light Sheet Fluorescence Microscopy}, journal = {Curr. Opin. Neurobiol.}, year = {2008}, volume = {18}, number = {6}, pages = {624--632}, url = {http://dx.doi.org/10.1016/j.conb.2009.03.008}, doi = {http://dx.doi.org/10.1016/j.conb.2009.03.008} }
Ketkova, L.A. and Churbanov, M.F. 3D laser ultramicroscopy: A method for nondestructive
characterization of micro- and nanoinclusions in high-purity
materials for fiber and power optics
2014 Inorg. Mater.
Vol. 50(12), pp. 1301-1316 
article DOI URL 
Abstract: This paper describes a 3D laser ultramicroscopy technique and
apparatus for the nondestructive characterization of heterophase
inclusions in bulk high-purity materials for fiber and power
optics. In this technique, the concentration and size of
inclusions undetectable by optical microscopy are determined
using CCD detection of the light scattered by them in a
direction normal to the incident laser beam at wavelengths from
0.63 to 0.98 $m. The detection limit of the technique in
terms of inclusion size is n (10--100) nm, the
range of detectable number concentrations is 1--1011 cm3, and
the scan depth in the sample is 1 cm. Its performance has been
evaluated using test systems. The potential of the technique is
illustrated by the dispersion analysis of promising materials
for IR fiber-optic applications.
BibTeX:
@article{Ketkova2014-sf,
  author = {Ketkova, L A and Churbanov, M F},
  title = {3D laser ultramicroscopy: A method for nondestructive
characterization of micro- and nanoinclusions in high-purity
materials for fiber and power optics}, journal = {Inorg. Mater.}, publisher = {Pleiades Publishing}, year = {2014}, volume = {50}, number = {12}, pages = {1301--1316}, url = {http://link.springer.com/article/10.1134/S0020168514120097}, doi = {http://dx.doi.org/10.1134/S0020168514120097} }
Khairy, K. and Keller, P.J. Reconstructing embryonic development 2011 Genesis
Vol. 49(7), pp. 488-513 
article DOI URL 
Abstract: Novel approaches to bio-imaging and automated computational
image processing allow the design of truly quantitative
studies in developmental biology. Cell behavior, cell fate
decisions, cell interactions during tissue morphogenesis, and
gene expression dynamics can be analyzed in vivo for entire
complex organisms and throughout embryonic development. We
review state-of-the-art technology for live imaging, focusing
on fluorescence light microscopy techniques for system-level
investigations of animal development, and discuss
computational approaches to image segmentation, cell tracking,
automated data annotation, and biophysical modeling. We argue
that the substantial increase in data complexity and size
requires sophisticated new strategies to data analysis to
exploit the enormous potential of these new resources.
BibTeX:
@article{Khairy2011-xx,
  author = {Khairy, Khaled and Keller, Philipp J},
  title = {Reconstructing embryonic development},
  journal = {Genesis},
  year = {2011},
  volume = {49},
  number = {7},
  pages = {488--513},
  url = {http://dx.doi.org/10.1002/dvg.20698},
  doi = {http://dx.doi.org/10.1002/dvg.20698}
}
Khairy, K., Lemon, W.C., Amat, F. and Keller, P.J. Light sheet-based imaging and analysis of early embryogenesis
in the fruit fly
2015 Methods Mol. Biol.
Vol. 1189, pp. 79-97 
article DOI URL 
Abstract: The fruit fly is an excellent model system for investigating
the sequence of epithelial tissue invaginations constituting
the process of gastrulation. By combining recent advancements
in light sheet fluorescence microscopy (LSFM) and image
processing, the three-dimensional fly embryo morphology and
relevant gene expression patterns can be accurately recorded
throughout the entire process of embryogenesis. LSFM provides
exceptionally high imaging speed, high signal-to-noise ratio,
low level of photoinduced damage, and good optical penetration
depth. This powerful combination of capabilities makes LSFM
particularly suitable for live imaging of the fly embryo.The
resulting high-information-content image data are subsequently
processed to obtain the outlines of cells and cell nuclei, as
well as the geometry of the whole embryo tissue by image
segmentation. Furthermore, morphodynamics information is
extracted by computationally tracking objects in the image.
Towards that goal we describe the successful implementation of
a fast fitting strategy of Gaussian mixture models.The data
obtained by image processing is well-suited for hypothesis
testing of the detailed biomechanics of the gastrulating
embryo. Typically this involves constructing computational
mechanics models that consist of an objective function
providing an estimate of strain energy for a given
morphological configuration of the tissue, and a numerical
minimization mechanism of this energy, achieved by varying
morphological parameters.In this chapter, we provide an
overview of in vivo imaging of fruit fly embryos using LSFM,
computational tools suitable for processing the resulting
images, and examples of computational biomechanical
simulations of fly embryo gastrulation.
BibTeX:
@article{Khairy2015-wf,
  author = {Khairy, Khaled and Lemon, William C and Amat, Fernando and
Keller, Philipp J}, title = {Light sheet-based imaging and analysis of early embryogenesis
in the fruit fly}, journal = {Methods Mol. Biol.}, year = {2015}, volume = {1189}, pages = {79--97}, url = {http://dx.doi.org/10.1007/978-1-4939-1164-6_6}, doi = {http://dx.doi.org/10.1007/978-1-4939-1164-6\_6} }
Kikuchi, S., Sonobe, K., Mashiko, S., Hiraoka, Y. and Ohyama, N. Three-dimensional image reconstruction for biological
micro-specimens using a double-axis fluorescence microscope
1997 Opt. Commun.
Vol. 138(1-3), pp. 21-26 
article DOI URL 
Abstract: A 3D imaging system using a double-axis fluorescence
microscope provides a practical way to reconstruct 3D images
of micro-objects from images simultaneously acquired from the
two orthogonal directions. In this paper, we show experimental
results of 3D image reconstruction using a water plant
specimen as an object.
BibTeX:
@article{Kikuchi1997-ba,
  author = {Kikuchi, Susumu and Sonobe, Kazuo and Mashiko, Shinro and
Hiraoka, Yasushi and Ohyama, Nagaaki}, title = {Three-dimensional image reconstruction for biological
micro-specimens using a double-axis fluorescence microscope}, journal = {Opt. Commun.}, year = {1997}, volume = {138}, number = {1--3}, pages = {21--26}, url = {http://www.sciencedirect.com/science/article/pii/S0030401897000540}, doi = {http://dx.doi.org/10.1016/S0030-4018(97)00054-0} }
Kikuchi, S., Sonobe, K., Sidharta, L.S. and Ohyama, N. Three-dimensional computed tomography for optical microscopes 1994 Opt. Commun.
Vol. 107(5), pp. 432-444 
article DOI URL 
Abstract: A 3D optical imaging method is presented based on computed
tomography techniques. The 3D optical transfer function of our
system has a higher band spherically, since 3D reconstructed
images are composed only of the in-focused information of
objects. Experimental results are shown for a simulated
fluorescence microscope.
BibTeX:
@article{Kikuchi1994-oa,
  author = {Kikuchi, Susumu and Sonobe, Kazuo and Sidharta, Liliek S and
Ohyama, Nagaaki}, title = {Three-dimensional computed tomography for optical microscopes}, journal = {Opt. Commun.}, year = {1994}, volume = {107}, number = {5}, pages = {432--444}, url = {http://www.sciencedirect.com/science/article/pii/0030401894903611}, doi = {http://dx.doi.org/10.1016/0030-4018(94)90361-1} }
Kirchmaier, B.C., Poon, K.L., Schwerte, T., Huisken, J., Winkler, C., Jungblut, B., Stainier, D.Y. and Brand, T. The Popeye domain containing 2 (popdc2) gene in zebrafish is
required for heart and skeletal muscle development
2012 Dev. Biol.
Vol. 363(2), pp. 438-450 
article DOI URL 
Abstract: The Popeye domain containing (Popdc) genes encode a family of
transmembrane proteins with an evolutionary conserved Popeye
domain. These genes are abundantly expressed in striated
muscle tissue, however their function is not well understood.
In this study we have investigated the role of the popdc2 gene
in zebrafish. Popdc2 transcripts were detected in the
embryonic myocardium and transiently in the craniofacial and
tail musculature. Morpholino oligonucleotide-mediated
knockdown of popdc2 resulted in aberrant development of
skeletal muscle and heart. Muscle segments in the trunk were
irregularly shaped and craniofacial muscles were severely
reduced or even missing. In the heart, pericardial edema was
prevalent in the morphants and heart chambers were elongated
and looping was abnormal. These pathologies in muscle and
heart were alleviated after reducing the morpholino
concentration. However the heart still was abnormal displaying
cardiac arrhythmia at later stages of development. Optical
recordings of cardiac contractility revealed irregular
ventricular contractions with a 2:1, or 3:1 atrial/ventricular
conduction ratio, which caused a significant reduction in
heart frequency. Recordings of calcium transients with high
spatiotemporal resolution using a transgenic calcium indicator
line (Tg(cmlc2:gCaMP)(s878)) and SPIM microscopy confirmed the
presence of a severe arrhythmia phenotype. Our results
identify popdc2 as a gene important for striated muscle
differentiation and cardiac morphogenesis. In addition it is
required for the development of the cardiac conduction system.
BibTeX:
@article{Kirchmaier2012-ls,
  author = {Kirchmaier, Bettina C and Poon, Kar Lai and Schwerte, Thorsten
and Huisken, Jan and Winkler, Christoph and Jungblut, Benno
and Stainier, Didier Y and Brand, Thomas}, title = {The Popeye domain containing 2 (popdc2) gene in zebrafish is
required for heart and skeletal muscle development}, journal = {Dev. Biol.}, year = {2012}, volume = {363}, number = {2}, pages = {438--450}, url = {http://dx.doi.org/10.1016/j.ydbio.2012.01.015}, doi = {http://dx.doi.org/10.1016/j.ydbio.2012.01.015} }
Klohs, J., Gräfe, M., Graf, K., Steinbrink, J., Dietrich, T., Stibenz, D., Bahmani, P., Kronenberg, G., Harms, C., Endres, M., Lindauer, U., Greger, K., Stelzer, E.H.K., Dirnagl, U. and Wunder, A. In vivo imaging of the inflammatory receptor CD40 after
cerebral ischemia using a fluorescent antibody
2008 Stroke
Vol. 39(10), pp. 2845-2852 
article DOI URL 
Abstract: BACKGROUND AND PURPOSE: Brain inflammation is a hallmark of
stroke, where it has been implicated in tissue damage as well
as in repair. Imaging technologies that specifically visualize
these processes are highly desirable. In this study, we
explored whether the inflammatory receptor CD40 can be
noninvasively and specifically visualized in mice after
cerebral ischemia using a fluorescent monoclonal antibody,
which we labeled with the near-infrared fluorescence dye Cy5.5
(Cy5.5-CD40MAb). METHODS: Wild-type and CD40-deficient mice
were subjected to transient middle cerebral artery occlusion.
Mice were either intravenously injected with Cy5.5-CD40MAb or
control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared
fluorescence imaging was performed after injection of the
compounds. Probe distribution and specificity was further
assessed with single-plane illumination microscopy,
immunohistochemistry, and confocal microscopy. RESULTS:
Significantly higher fluorescence intensities over the
stroke-affected hemisphere, compared to the contralateral
side, were only detected noninvasively in wild-type mice that
received Cy5.5-CD40MAb, but not in CD40-deficient mice
injected with Cy5.5-CD40MAb or in wild-type mice that were
injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence
showed an intense fluorescence within the ischemic territory
only in wild-type mice injected with Cy5.5-CD40MAb. In the
brains of these mice, single-plane illumination microscopy
demonstrated vascular and parenchymal distribution, and
confocal microscopy revealed a partial colocalization of
parenchymal fluorescence from the injected Cy5.5-CD40MAb with
activated microglia and blood-derived cells in the ischemic
region. CONCLUSIONS: The study demonstrates that a
CD40-targeted fluorescent antibody enables specific
noninvasive detection of the inflammatory receptor CD40 after
cerebral ischemia using optical techniques.
BibTeX:
@article{Klohs2008-dc,
  author = {Klohs, Jan and Gräfe, Michael and Graf, Kristof and
Steinbrink, Jens and Dietrich, Thore and Stibenz, Dietger and
Bahmani, Peyman and Kronenberg, Golo and Harms, Christoph and
Endres, Matthias and Lindauer, Ute and Greger, Klaus and
Stelzer, Ernst H K and Dirnagl, Ulrich and Wunder, Andreas}, title = {In vivo imaging of the inflammatory receptor CD40 after
cerebral ischemia using a fluorescent antibody}, journal = {Stroke}, year = {2008}, volume = {39}, number = {10}, pages = {2845--2852}, url = {http://dx.doi.org/10.1161/STROKEAHA.107.509844}, doi = {http://dx.doi.org/10.1161/STROKEAHA.107.509844} }
Kobitski, A.Y., Otte, J.C., Takamiya, M., Schäfer, B., Mertes, J., Stegmaier, J., Rastegar, S., Rindone, F., Hartmann, V., Stotzka, R., Garca, A., van Wezel, J., Mikut, R., Strähle, U. and Nienhaus, G.U. An ensemble-averaged, cell density-based digital model of
zebrafish embryo development derived from light-sheet
microscopy data with single-cell resolution
2015 Sci. Rep.
Vol. 5, pp. 8601 
article DOI URL 
Abstract: A new era in developmental biology has been ushered in by
recent advances in the quantitative imaging of all-cell
morphogenesis in living organisms. Here we have developed a
light-sheet fluorescence microscopy-based framework with
single-cell resolution for identification and characterization
of subtle phenotypical changes of millimeter-sized organisms.
Such a comparative study requires analyses of entire ensembles
to be able to distinguish sample-to-sample variations from
definitive phenotypical changes. We present a kinetic digital
model of zebrafish embryos up to 16 h of development. The
model is based on the precise overlay and averaging of data
taken on multiple individuals and describes the cell density
and its migration direction at every point in time.
Quantitative metrics for multi-sample comparative studies have
been introduced to analyze developmental variations within the
ensemble. The digital model may serve as a canvas on which the
behavior of cellular subpopulations can be studied. As an
example, we have investigated cellular rearrangements during
germ layer formation at the onset of gastrulation. A
comparison of the one-eyed pinhead (oep) mutant with the
digital model of the wild-type embryo reveals its abnormal
development at the onset of gastrulation, many hours before
changes are obvious to the eye.
BibTeX:
@article{Kobitski2015-op,
  author = {Kobitski, Andrei Y and Otte, Jens C and Takamiya, Masanari and
Schäfer, Benjamin and Mertes, Jonas and Stegmaier,
Johannes and Rastegar, Sepand and Rindone, Francesca and
Hartmann, Volker and Stotzka, Rainer and Garca, Ariel
and van Wezel, Jos and Mikut, Ralf and Strähle, Uwe and
Nienhaus, G Ulrich}, title = {An ensemble-averaged, cell density-based digital model of
zebrafish embryo development derived from light-sheet
microscopy data with single-cell resolution}, journal = {Sci. Rep.}, year = {2015}, volume = {5}, pages = {8601}, url = {http://dx.doi.org/10.1038/srep08601}, doi = {http://dx.doi.org/10.1038/srep08601} }
Konopka, C.A. and Bednarek, S.Y. Variable-angle epifluorescence microscopy: a new way to look
at protein dynamics in the plant cell cortex
2008 Plant J.
Vol. 53(1), pp. 186-196 
article DOI URL 
Abstract: Live-cell microscopy imaging of fluorescent-tagged fusion
proteins is an essential tool for cell biologists. Total
internal reflection fluorescence microscopy (TIRFM) has joined
confocal microscopy as a complementary system for the imaging
of cell surface protein dynamics in mammalian and yeast
systems because of its high temporal and spatial resolution.
Here we present an alternative to TIRFM, termed variable-angle
epifluorescence microscopy (VAEM), for the visualization of
protein dynamics at or near the plasma membrane of plant
epidermal cells and root hairs in whole, intact seedlings that
provides high-signal, low-background and near real-time
imaging. VAEM uses highly oblique subcritical incident angles
to decrease background fluorophore excitation. We discuss the
utilities and advantages of VAEM for imaging of fluorescent
fusion-tagged marker proteins in studying cortical
cytoskeletal and membrane proteins. We believe that the
application of VAEM will be an invaluable imaging tool for
plant cell biologists.
BibTeX:
@article{Konopka2008-dn,
  author = {Konopka, Catherine A and Bednarek, Sebastian Y},
  title = {Variable-angle epifluorescence microscopy: a new way to look
at protein dynamics in the plant cell cortex}, journal = {Plant J.}, year = {2008}, volume = {53}, number = {1}, pages = {186--196}, url = {http://dx.doi.org/10.1111/j.1365-313X.2007.03306.x}, doi = {http://dx.doi.org/10.1111/j.1365-313X.2007.03306.x} }
Kozubek, M., Skalnková, M., Matula, P., Bártová, E., Rauch, J., Neuhaus, F., Eipel, H. and Hausmann, M. Automated microaxial tomography of cell nuclei after specific
labelling by fluorescence in situ hybridisation
2002 Micron
Vol. 33(7-8), pp. 655-665 
article URL 
Abstract: Microaxial tomography provides a good means for microscopic
image acquisition of cells or sub-cellular components like
cell nuclei with an improved resolution, because shortcomings
of spatial resolution anisotropy in optical microscopy can be
overcome. Thus, spatial information of the object can be
obtained without the necessity of confocal imaging. Since the
very early developments of microaxial tomography, a
considerable drawback of this method was a complicated image
acquisition and processing procedure that requires much
operator time. In order to solve this problem the Heidelberg
2pi-tilting device has been mounted on the Brno
high-resolution cytometer as an attempt to bring together
advanced microscopy and fast automated computer image
acquisition and analysis. A special software module that
drives all hardware components required for automated
microaxial tomography and performs image acquisition and
processing has been developed. First, a general image
acquisition strategy is presented. Then the procedure for
automation of axial tomography and the developed software
module are described. The rotation precision has been
experimentally proved followed by experiments with a specific
biological example. For this application, also a method for
the preparation of cell nuclei attached to glass fibres has
been developed that allows for the first time imaging of
three-dimensionally conserved, fluorescence in situ
hybridisation-stained cell nuclei fixed to a glass fibre.
BibTeX:
@article{Kozubek2002-ke,
  author = {Kozubek, M and Skalnková, M and Matula, Pe and
Bártová, E and Rauch, J and Neuhaus, F and Eipel, H
and Hausmann, M}, title = {Automated microaxial tomography of cell nuclei after specific
labelling by fluorescence in situ hybridisation}, journal = {Micron}, year = {2002}, volume = {33}, number = {7-8}, pages = {655--665}, url = {http://www.ncbi.nlm.nih.gov/pubmed/12475562} }
Krzic, U. Multiple-view microscopy with light-sheet based fluorescence
microscope
2009   phdthesis URL 
Abstract: The axial resolution of any standard single-lens light microscope
is lower than its lateral resolution. The ratio is approximately
3-4 when high numerical aperture objective lenses are used (NA
1.2 -1.4) and more than 10 with low numerical apertures (NA 0.2
and below). In biological imaging, the axial resolution is
normally insufficient to resolve subcellular phenomena.
Furthermore, parts of the images of opaque specimens are often
highly degraded or obscured. Multiple-view fluorescence
microscopy overcomes both problems simultaneously by recording
multiple images of the same specimen along different directions.
The images are digitally fused into a single high-quality image.
Multiple-view imaging was developed as an extension to the
light-sheet based fluorescence microscope (LSFM), a novel
technique that seems to be better suited for multiple-view
imaging than any other fluorescence microscopy method to date. In
this contribution, the LSFM properties, which are important for
multiple-view imaging, are characterized and the implementation
of LSFM based multiple-view microscopy is described. The
important aspects of multiple-view image alignment and fusion are
discussed, the published algorithms are reviewed and original
solutions are proposed. The advantages and limitations of
multiple-view imaging with LSFM are demonstrated using a number
of specimens, which range in size from a single yeast cell to an
adult fruit fly and to Medaka fish.
BibTeX:
@phdthesis{Krzic2009-yn,
  author = {Krzic, Uros},
  title = {Multiple-view microscopy with light-sheet based fluorescence
microscope}, year = {2009}, url = {http://archiv.ub.uni-heidelberg.de/volltextserver/9668/} }
Krzic, U., Gunther, S., Saunders, T.E., Streichan, S.J. and Hufnagel, L. Multiview light-sheet microscope for rapid in toto imaging 2012 Nat. Methods
Vol. 9(7), pp. 730-733 
article DOI URL 
Abstract: We present a multiview selective-plane illumination microscope
(MuVi-SPIM), comprising two detection and illumination
objective lenses, that allows rapid in toto fluorescence
imaging of biological specimens with subcellular resolution.
The fixed geometrical arrangement of the imaging branches
enables multiview data fusion in real time. The high speed of
MuVi-SPIM allows faithful tracking of nuclei and cell shape
changes, which we demonstrate through in toto imaging of the
embryonic development of Drosophila melanogaster.
BibTeX:
@article{Krzic2012-nr,
  author = {Krzic, Uros and Gunther, Stefan and Saunders, Timothy E and
Streichan, Sebastian J and Hufnagel, Lars}, title = {Multiview light-sheet microscope for rapid in toto imaging}, journal = {Nat. Methods}, publisher = {Nature Publishing Group}, year = {2012}, volume = {9}, number = {7}, pages = {730--733}, url = {http://dx.doi.org/10.1038/nmeth.2064}, doi = {http://dx.doi.org/10.1038/nmeth.2064} }
Kumar, A., Colón-Ramos, D.A. and Shroff, H. Watching a roundworm develop with a sheet of light 2015 Phys. Today
Vol. 68(7), pp. 58-59 
article DOI URL 
Abstract: Light-sheet fluorescence microscopy allows neurobiologists to
observe fundamental biological processes in real time.
BibTeX:
@article{Kumar2015-fl,
  author = {Kumar, Abhishek and Colón-Ramos, Daniel A and Shroff, Hari},
  title = {Watching a roundworm develop with a sheet of light},
  journal = {Phys. Today},
  publisher = {American Institute of Physics},
  year = {2015},
  volume = {68},
  number = {7},
  pages = {58--59},
  url = {http://scitation.aip.org/content/aip/magazine/physicstoday/article/68/7/10.1063/PT.3.2856},
  doi = {http://dx.doi.org/10.1063/PT.3.2856}
}
Kumar, A., Wu, Y., Christensen, R., Chandris, P., Gandler, W., McCreedy, E., Bokinsky, A., Colón-Ramos, D.A., Bao, Z., McAuliffe, M., Rondeau, G. and Shroff, H. Dual-view plane illumination microscopy for rapid and
spatially isotropic imaging
2014 Nat. Protoc.
Vol. 9(11), pp. 2555-2573 
article DOI URL 
Abstract: We describe the construction and use of a compact dual-view
inverted selective plane illumination microscope (diSPIM) for
time-lapse volumetric (4D) imaging of living samples at
subcellular resolution. Our protocol enables a biologist with
some prior microscopy experience to assemble a diSPIM from
commercially available parts, to align optics and test system
performance, to prepare samples, and to control hardware and
data processing with our software. Unlike existing light sheet
microscopy protocols, our method does not require the sample
to be embedded in agarose; instead, samples are prepared
conventionally on glass coverslips. Tissue culture cells and
Caenorhabditis elegans embryos are used as examples in this
protocol; successful implementation of the protocol results in
isotropic resolution and acquisition speeds up to several
volumes per s on these samples. Assembling and verifying
diSPIM performance takes 6 d, sample preparation and data
acquisition take up to 5 d and postprocessing takes 3-8 h,
depending on the size of the data.
BibTeX:
@article{Kumar2014-qm,
  author = {Kumar, Abhishek and Wu, Yicong and Christensen, Ryan and
Chandris, Panagiotis and Gandler, William and McCreedy, Evan
and Bokinsky, Alexandra and Colón-Ramos, Daniel A and Bao,
Zhirong and McAuliffe, Matthew and Rondeau, Gary and Shroff,
Hari}, title = {Dual-view plane illumination microscopy for rapid and
spatially isotropic imaging}, journal = {Nat. Protoc.}, publisher = {Nature Publishing Group}, year = {2014}, volume = {9}, number = {11}, pages = {2555--2573}, url = {http://dx.doi.org/10.1038/nprot.2014.172}, doi = {http://dx.doi.org/10.1038/nprot.2014.172} }
Kumar, S., Wilding, D., Sikkel, M.B., Lyon, A.R., MacLeod, K.T. and Dunsby, C. Application of oblique plane microscopy to high speed live cell
imaging
2011 European Conference on Biomedical Optics, pp. 80860V  inproceedings DOI URL 
Abstract: Oblique Plane Microscopy (OPM) is a light sheet microscopy
technique that combines oblique illumination with correction
optics that tilt the focal plane of the collection system. OPM
can be used to image conventionally mounted specimens on
coverslips or tissue culture dishes and has low out-of-plane
photobleaching and phototoxicity. No moving parts are required
to achieve an optically sectioned image and so high speed
optically sectioned imaging is possible. We present high speed
2D and 3D optically sectioned OPM imaging of live cells using a
high NA water immersion lens.
BibTeX:
@inproceedings{Kumar2011-dq,
  author = {Kumar, Sunil and Wilding, Dean and Sikkel, Markus B and Lyon,
Alexander R and MacLeod, Ken T and Dunsby, Chris}, title = {Application of oblique plane microscopy to high speed live cell
imaging}, booktitle = {European Conference on Biomedical Optics}, publisher = {Optical Society of America}, year = {2011}, pages = {80860V}, url = {http://www.opticsinfobase.org/abstract.cfm?URI=ECBO-2011-80860V}, doi = {http://dx.doi.org/10.1364/ECBO.2011.80860V} }
Kumar, S., Wilding, D., Sikkel, M.B., Lyon, A.R., MacLeod, K.T. and Dunsby, C. High-speed 2D and 3D fluorescence microscopy of cardiac
myocytes
2011 Opt. Express
Vol. 19(15), pp. 13839-13847 
article DOI URL 
Abstract: Oblique plane microscopy (OPM) is a light sheet microscopy
technique that uses a single high numerical aperture
microscope objective to both illuminate a tilted plane within
the specimen and to obtain an image of the tilted illuminated
plane. In this paper, we present a new OPM configuration that
enables both the illumination and detection focal planes to be
swept simultaneously and remotely through the sample volume,
enabling high speed volumetric imaging. We demonstrate the
high speed imaging capabilities of the system by imaging
calcium dynamics in cardiac myocytes in 2D at 926 frames per
second and in 3D at 21 volumes per second. In the future,
higher frame rate CCD cameras will enable volumetric imaging
at much greater rates, leading to new capabilities to study
dynamic events in cells at high speeds in two and three
dimensions.
BibTeX:
@article{Kumar2011-ot,
  author = {Kumar, Sunil and Wilding, Dean and Sikkel, Markus B and Lyon,
Alexander R and MacLeod, Ken T and Dunsby, Chris}, title = {High-speed 2D and 3D fluorescence microscopy of cardiac
myocytes}, journal = {Opt. Express}, year = {2011}, volume = {19}, number = {15}, pages = {13839--13847}, url = {http://dx.doi.org/10.1364/OE.19.013839}, doi = {http://dx.doi.org/10.1364/OE.19.013839} }
Laksameethanasan, D., Brandt, S.S., Engelhardt, P., Renaud, O. and Shorte, S.L. A Bayesian reconstruction method for micro-rotation imaging in
light microscopy
2008 Microsc. Res. Tech.
Vol. 71(2), pp. 158-167 
article DOI URL 
Abstract: The authors present a three-dimensional (3D) reconstruction
algorithm and reconstruction-based deblurring method for light
microscopy using a micro-rotation device. In contrast to
conventional 3D optical imaging where the focal plane is
shifted along the optical axis, micro-rotation imaging employs
dielectric fields to rotate the object inside a fixed optical
set-up. To address this entirely new 3D-imaging modality, the
authors present a reconstruction algorithm based on Bayesian
inversion theory and use the total variation function as a
structure prior. The spectral properties of the reconstruction
by simulations that illustrate the strengths and the
weaknesses of the micro-rotation approach, compared with
conventional 3D optical imaging, were studied. The
reconstruction from real data sets shows that this method is
promising for 3D reconstruction and offers itself as a
deblurring method using a reconstruction-based procedure for
removing out-of-focus light from the micro-rotation image
series.
BibTeX:
@article{Laksameethanasan2008-af,
  author = {Laksameethanasan, Danai and Brandt, Sami S and Engelhardt,
Peter and Renaud, Olivier and Shorte, Spencer L}, title = {A Bayesian reconstruction method for micro-rotation imaging in
light microscopy}, journal = {Microsc. Res. Tech.}, year = {2008}, volume = {71}, number = {2}, pages = {158--167}, url = {http://dx.doi.org/10.1002/jemt.20550}, doi = {http://dx.doi.org/10.1002/jemt.20550} }
Langhans, M. and Meckel, T. Single-molecule detection and tracking in plants 2014 Protoplasma
Vol. 251(2), pp. 277-291 
article DOI URL 
Abstract: Combining optical properties with a limited choice of
fluorophores turns single-molecule imaging in plants into a
challenging task. This explains why the technique, despite its
success in the field of animal cell biology, is far from being
routinely applied in plant cell research. The same challenges,
however, also apply to the application of single-molecule
microscopy to any intact tissue or multicellular 3D cell
culture. As recent and upcoming progress in fluorescence
microscopy will permit single-molecule detection in the
context of multicellular systems, plant tissue imaging will
experience a huge benefit from this progress. In this review,
we address every step of a single-molecule experiment,
highlight the critical aspects of each and elaborate on
optimizations and developments required for improvements. We
relate each step to recent achievements, which have so far
been conducted exclusively on the root epidermis of
Arabidopsis thaliana seedlings with inclined illumination and
show examples of single-molecule measurements using different
cells or illumination schemes.
BibTeX:
@article{Langhans2014-kr,
  author = {Langhans, Markus and Meckel, Tobias},
  title = {Single-molecule detection and tracking in plants},
  journal = {Protoplasma},
  year = {2014},
  volume = {251},
  number = {2},
  pages = {277--291},
  url = {http://dx.doi.org/10.1007/s00709-013-0601-0},
  doi = {http://dx.doi.org/10.1007/s00709-013-0601-0}
}
Lauri, A., Brunet, T., Handberg-Thorsager, M., Fischer, A.H.L., Simakov, O., Steinmetz, P.R.H., Tomer, R., Keller, P.J. and Arendt, D. Development of the annelid axochord: insights into notochord
evolution
2014 Science
Vol. 345(6202), pp. 1365-1368 
article DOI URL 
Abstract: The origin of chordates has been debated for more than a
century, with one key issue being the emergence of the
notochord. In vertebrates, the notochord develops by
convergence and extension of the chordamesoderm, a population
of midline cells of unique molecular identity. We identify a
population of mesodermal cells in a developing invertebrate,
the marine annelid Platynereis dumerilii, that converges and
extends toward the midline and expresses a notochord-specific
combination of genes. These cells differentiate into a
longitudinal muscle, the axochord, that is positioned between
central nervous system and axial blood vessel and secretes a
strong collagenous extracellular matrix. Ancestral state
reconstruction suggests that contractile mesodermal midline
cells existed in bilaterian ancestors. We propose that these
cells, via vacuolization and stiffening, gave rise to the
chordate notochord.
BibTeX:
@article{Lauri2014-dh,
  author = {Lauri, Antonella and Brunet, Thibaut and Handberg-Thorsager,
Mette and Fischer, Antje H L and Simakov, Oleg and Steinmetz,
Patrick R H and Tomer, Raju and Keller, Philipp J and Arendt,
Detlev}, title = {Development of the annelid axochord: insights into notochord
evolution}, journal = {Science}, year = {2014}, volume = {345}, number = {6202}, pages = {1365--1368}, url = {http://dx.doi.org/10.1126/science.1253396}, doi = {http://dx.doi.org/10.1126/science.1253396} }
Lavagnino, Z., Zanacchi, F.C., Ronzitti, E. and Diaspro, A. Two-photon excitation selective plane illumination microscopy
(2PE-SPIM) of highly scattering samples: characterization
and application
2013 Opt. Express
Vol. 21(5), pp. 5998-6008 
article DOI URL 
Abstract: In this work we report the advantages provided by two photon
excitation (2PE) implemented in a selective plane illumination
microscopy (SPIM) when imaging thick scattering samples. In
particular, a detailed analysis of the effects induced on the
real light sheet excitation intensity distribution is
performed. The comparison between single-photon and two-photon
excitation profiles shows the reduction of the scattering
effects and sample-induced aberrations provided by 2PE-SPIM.
Furthermore, uniformity of the excitation distribution and the
consequent improved image contrast is shown when imaging
scattering phantom samples in depth by 2PE-SPIM. These results
show the advantages of 2PE-SPIM and suggest how this
combination can further enhance the SPIM performance. Phantom
samples have been designed with optical properties compatible
with biological applications of interest.
BibTeX:
@article{Lavagnino2013-un,
  author = {Lavagnino, Zeno and Zanacchi, Francesca Cella and Ronzitti,
Emiliano and Diaspro, Alberto}, title = {Two-photon excitation selective plane illumination microscopy
(2PE-SPIM) of highly scattering samples: characterization
and application}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {5}, pages = {5998--6008}, url = {http://dx.doi.org/10.1364/OE.21.005998}, doi = {http://dx.doi.org/10.1364/OE.21.005998} }
Lee, H.K., Uddin, M.S., Sankaran, S., Hariharan, S. and Ahmed, S. A field theoretical restoration method for images degraded by
non-uniform light attenuation : an application for light
microscopy
2009 Opt. Express
Vol. 17(14), pp. 11294-11308 
article URL 
Abstract: Microscopy has become a de facto tool for biology. However, it
suffers from a fundamental problem of poor contrast with
increasing depth, as the illuminating light gets attenuated
and scattered and hence can not penetrate through thick
samples. The resulting decay of light intensity due to
attenuation and scattering varies exponentially across the
image. The classical space invariant deconvolution approaches
alone are not suitable for the restoration of uneven
illumination in microscopy images. In this paper, we present a
novel physics-based field theoretical approach to solve the
contrast degradation problem of light microscopy images. We
have confirmed the effectiveness of our technique through
simulations as well as through real field experimentations.
BibTeX:
@article{Lee2009-yg,
  author = {Lee, Hwee Kuan and Uddin, Mohammad Shorif and Sankaran,
Shvetha and Hariharan, Srivats and Ahmed, Sohail}, title = {A field theoretical restoration method for images degraded by
non-uniform light attenuation : an application for light
microscopy}, journal = {Opt. Express}, year = {2009}, volume = {17}, number = {14}, pages = {11294--11308}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19582043} }
Lei, M. and Zumbusch, A. Structured light sheet fluorescence microscopy based on four
beam interference
2010 Opt. Express
Vol. 18(18), pp. 19232-19241 
article DOI URL 
Abstract: A 3D structured light sheet microscope using a four-faceted
symmetric pyramid is presented. The sample is illuminated by
the resulting four beam interference field. This approach
combines advantages of standing wave and structured
illumination microscopy. Examples of micrographs of
fluorescently labeled Chinese hamster ovary (CHO) cells as
well as of the compound eyes of drosophila are shown and the
optical sectioning ability of our system is demonstrated. The
capabilities and the limitations of the scheme are discussed.
BibTeX:
@article{Lei2010-vo,
  author = {Lei, Ming and Zumbusch, Andreas},
  title = {Structured light sheet fluorescence microscopy based on four
beam interference}, journal = {Opt. Express}, year = {2010}, volume = {18}, number = {18}, pages = {19232--19241}, url = {http://dx.doi.org/10.1364/OE.18.019232}, doi = {http://dx.doi.org/10.1364/OE.18.019232} }
Leischner, U., Schierloh, A., Zieglgänsberger, W. and Dodt, H.-U. Formalin-induced fluorescence reveals cell shape and
morphology in biological tissue samples
2010 PLoS One
Vol. 5(4), pp. e10391 
article DOI URL 
Abstract: Ultramicroscopy is a powerful tool to reveal detailed
three-dimensional structures of large microscopical objects.
Using high magnification, we observed that formalin induces
fluorescence more in extra-cellular space and stains cellular
structures negatively, rendering cells as dark objects in
front of a bright background. Here, we show this effect on a
three-dimensional image stack of a hippocampus sample,
focusing on the CA1 region. This method, called
FIF-Ultramicroscopy, allows for the three-dimensional
observation of cellular structures in various tissue types
without complicated staining techniques.
BibTeX:
@article{Leischner2010-db,
  author = {Leischner, Ulrich and Schierloh, Anja and
Zieglgänsberger, Walter and Dodt, Hans-Ulrich}, title = {Formalin-induced fluorescence reveals cell shape and
morphology in biological tissue samples}, journal = {PLoS One}, year = {2010}, volume = {5}, number = {4}, pages = {e10391}, url = {http://dx.doi.org/10.1371/journal.pone.0010391}, doi = {http://dx.doi.org/10.1371/journal.pone.0010391} }
Leischner, U., Zieglgänsberger, W. and Dodt, H.-U. Resolution of ultramicroscopy and field of view analysis 2009 PLoS One
Vol. 4(6), pp. e5785 
article DOI URL 
Abstract: In a recent publication we described a microscopical technique
called Ultramicroscopy, combined with a histological procedure
that makes biological samples transparent. With this
combination we can gather three-dimensional image data of
large biological samples. Here we present the theoretical
analysis of the z-resolution. By analyzing the cross-section
of the illuminating sheet of light we derive resolution values
according to the Rayleigh-criterion. Next we investigate the
resolution adjacent to the focal point of the illumination
beam, analyze throughout what extend the illumination beam is
of acceptable sharpness and investigate the resolution
improvements caused by the objective lens. Finally we conclude
with a useful rule for the sampling rates. These findings are
of practical importance for researchers working with
Ultramicroscopy to decide on adequate sampling rates. They are
also necessary to modify deconvolution techniques to gain
further image improvements.
BibTeX:
@article{Leischner2009-pc,
  author = {Leischner, Ulrich and Zieglgänsberger, Walter and Dodt,
Hans-Ulrich}, title = {Resolution of ultramicroscopy and field of view analysis}, journal = {PLoS One}, year = {2009}, volume = {4}, number = {6}, pages = {e5785}, url = {http://dx.doi.org/10.1371/journal.pone.0005785}, doi = {http://dx.doi.org/10.1371/journal.pone.0005785} }
Lemon, W.C. and Keller, P.J. Live imaging of nervous system development and function using
light-sheet microscopy
2015 Mol. Reprod. Dev.
Vol. 82(7-8), pp. 605-618 
article DOI URL 
Abstract: In vivo imaging applications typically require carefully
balancing conflicting parameters. Often it is necessary to
achieve high imaging speed, low photo-bleaching, and
photo-toxicity, good three-dimensional resolution, high
signal-to-noise ratio, and excellent physical coverage at the
same time. Light-sheet microscopy provides good performance in
all of these categories, and is thus emerging as a
particularly powerful live imaging method for the life
sciences. We see an outstanding potential for applying
light-sheet microscopy to the study of development and
function of the early nervous system in vertebrates and higher
invertebrates. Here, we review state-of-the-art approaches to
live imaging of early development, and show how the unique
capabilities of light-sheet microscopy can further advance our
understanding of the development and function of the nervous
system. We discuss key considerations in the design of
light-sheet microscopy experiments, including sample
preparation and fluorescent marker strategies, and provide an
outlook for future directions in the field. Mol. Reprod. Dev.
82: 605-618, 2015. pyright 2013 Wiley Periodicals, Inc.
BibTeX:
@article{Lemon2015-vs,
  author = {Lemon, William C and Keller, Philipp J},
  title = {Live imaging of nervous system development and function using
light-sheet microscopy}, journal = {Mol. Reprod. Dev.}, year = {2015}, volume = {82}, number = {7-8}, pages = {605--618}, url = {http://dx.doi.org/10.1002/mrd.22258}, doi = {http://dx.doi.org/10.1002/mrd.22258} }
Lemon, W.C., Pulver, S.R., Höckendorf, B., McDole, K., Branson, K., Freeman, J. and Keller, P.J. Whole-central nervous system functional imaging in larval
Drosophila
2015 Nat. Commun.
Vol. 6, pp. 7924 
article DOI URL 
Abstract: Understanding how the brain works in tight concert with the
rest of the central nervous system (CNS) hinges upon knowledge
of coordinated activity patterns across the whole CNS. We
present a method for measuring activity in an entire,
non-transparent CNS with high spatiotemporal resolution. We
combine a light-sheet microscope capable of simultaneous
multi-view imaging at volumetric speeds 25-fold faster than
the state-of-the-art, a whole-CNS imaging assay for the
isolated Drosophila larval CNS and a computational framework
for analysing multi-view, whole-CNS calcium imaging data. We
image both brain and ventral nerve cord, covering the entire
CNS at 2 or 5 Hz with two- or one-photon excitation,
respectively. By mapping network activity during fictive
behaviours and quantitatively comparing high-resolution
whole-CNS activity maps across individuals, we predict
functional connections between CNS regions and reveal neurons
in the brain that identify type and temporal state of motor
programs executed in the ventral nerve cord.
BibTeX:
@article{Lemon2015-hk,
  author = {Lemon, William C and Pulver, Stefan R and Höckendorf,
Burkhard and McDole, Katie and Branson, Kristin and Freeman,
Jeremy and Keller, Philipp J}, title = {Whole-central nervous system functional imaging in larval
Drosophila}, journal = {Nat. Commun.}, year = {2015}, volume = {6}, pages = {7924}, url = {http://dx.doi.org/10.1038/ncomms8924}, doi = {http://dx.doi.org/10.1038/ncomms8924} }
Lenard, A., Daetwyler, S., Betz, C., Ellertsdottir, E., Belting, H.-G., Huisken, J. and Affolter, M. Endothelial cell self-fusion during vascular pruning 2015 PLoS Biol.
Vol. 13(4), pp. e1002126 
article DOI URL 
Abstract: During embryonic development, vascular networks remodel to
meet the increasing demand of growing tissues for oxygen and
nutrients. This is achieved by the pruning of redundant blood
vessel segments, which then allows more efficient blood flow
patterns. Because of the lack of an in vivo system suitable
for high-resolution live imaging, the dynamics of the pruning
process have not been described in detail. Here, we present
the subintestinal vein (SIV) plexus of the zebrafish embryo as
a novel model to study pruning at the cellular level. We show
that blood vessel regression is a coordinated process of cell
rearrangements involving lumen collapse and cell-cell contact
resolution. Interestingly, the cellular rearrangements during
pruning resemble endothelial cell behavior during vessel
fusion in a reversed order. In pruning segments, endothelial
cells first migrate toward opposing sides where they join the
parental vascular branches, thus remodeling the multicellular
segment into a unicellular connection. Often, the lumen is
maintained throughout this process, and transient unicellular
tubes form through cell self-fusion. In a second step, the
unicellular connection is resolved unilaterally, and the
pruning cell rejoins the opposing branch. Thus, we show for
the first time that various cellular activities are
coordinated to achieve blood vessel pruning and define two
different morphogenetic pathways, which are selected by the
flow environment.
BibTeX:
@article{Lenard2015-nd,
  author = {Lenard, Anna and Daetwyler, Stephan and Betz, Charles and
Ellertsdottir, Elin and Belting, Heinz-Georg and Huisken, Jan
and Affolter, Markus}, title = {Endothelial cell self-fusion during vascular pruning}, journal = {PLoS Biol.}, year = {2015}, volume = {13}, number = {4}, pages = {e1002126}, url = {http://dx.doi.org/10.1371/journal.pbio.1002126}, doi = {http://dx.doi.org/10.1371/journal.pbio.1002126} }
Li, R., Zhou, X., Wu, D., Peng, T., Yang, Y., Lei, M., Yu, X., Yao, B. and Ye, T. Selective plane illumination microscopy with structured
illumination based on spatial light modulators
2014 SPIE BiOS, pp. 89491S-89491S-5  inproceedings DOI URL 
BibTeX:
@inproceedings{Li2014-zy,
  author = {Li, Runze and Zhou, Xing and Wu, Di and Peng, Tong and Yang,
Yanlong and Lei, Ming and Yu, Xianhua and Yao, Baoli and Ye,
Tong}, title = {Selective plane illumination microscopy with structured
illumination based on spatial light modulators}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {89491S--89491S--5}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1848542}, doi = {http://dx.doi.org/10.1117/12.2040319} }
Li, Y., Hu, Y. and Cang, H. Light sheet microscopy for tracking single molecules on the
apical surface of living cells
2013 J. Phys. Chem. B
Vol. 117(49), pp. 15503-15511 
article DOI URL 
Abstract: Single particle tracking is a powerful tool to study single
molecule dynamics in living biological samples. However,
current tracking techniques, which are based mainly on
epifluorescence, confocal, or TIRF microscopy, have
difficulties in tracking single molecules on the apical
surface of a cell. We present here a three-dimensional (3D)
single particle tracking technique that is based on prism
coupled light-sheet microscopy (PCLSM). This novel design
provides a signal-to-noise ratio comparable to confocal
microscopy while it has the capability of illuminating at
arbitrary depth. We demonstrate tracking of single EGF
molcules on the apical surface of live cell membranes from
their binding to EGF receptors until they are internalized or
photobleached. We found that EGF exhibits multiple diffusion
behaviors on live A549 cell membranes. At room temperature,
the average diffusion coefficient of EGF on A549 cells was
measured to be 0.13 $m(2)/s. Depletion of cellular
cholesterol with methyl-$-cyclodextrin leads to a
broader distribution of diffusion coefficients and an increase
of the average diffusion coefficient at room temperature. This
light-sheet based 3D single particle tracking technique solves
the technique difficulty of tracking single particles on
apical membranes and is able to document the whole
``lifetime'' of a particle from binding till photobleaching or
internalization.
BibTeX:
@article{Li2013-th,
  author = {Li, Yu and Hu, Ying and Cang, Hu},
  title = {Light sheet microscopy for tracking single molecules on the
apical surface of living cells}, journal = {J. Phys. Chem. B}, year = {2013}, volume = {117}, number = {49}, pages = {15503--15511}, url = {http://dx.doi.org/10.1021/jp405380g}, doi = {http://dx.doi.org/10.1021/jp405380g} }
Lim, J., Lee, H.K., Yu, W. and Ahmed, S. Light sheet fluorescence microscopy (LSFM): past, present and
future
2014 Analyst
Vol. 139(19), pp. 4758-4768 
article DOI URL 
Abstract: Light sheet fluorescence microscopy (LSFM) has emerged as an
important imaging modality to follow biology in live 3D samples
over time with reduced phototoxicity and photobleaching. In
particular, LSFM has been instrumental in revealing the detail
of early embryonic development of Zebrafish, Drosophila, and C.
elegans. Open access projects, DIY-SPIM, OpenSPIM, and OpenSPIN,
now allow LSFM to be set-up easily and at low cost. The aim of
this paper is to facilitate the set-up and use of LSFM by
reviewing and comparing open access projects, image processing
tools and future challenges.
BibTeX:
@article{Lim2014-mq,
  author = {Lim, John and Lee, Hwee Kuan and Yu, Weimiao and Ahmed, Sohail},
  title = {Light sheet fluorescence microscopy (LSFM): past, present and
future}, journal = {Analyst}, publisher = {The Royal Society of Chemistry}, year = {2014}, volume = {139}, number = {19}, pages = {4758--4768}, url = {http://pubs.rsc.org/en/Content/ArticleLanding/2014/AN/C4AN00624K}, doi = {http://dx.doi.org/10.1039/C4AN00624K} }
Lin, H.-C.A., Chekkoury, A., Omar, M., Schmitt-Manderbach, T., Koberstein-Schwarz, B., Mappes, T., López-Schier, H., Razansky, D. and Ntziachristos, V. Selective plane illumination optical and optoacoustic microscopy
for postembryonic imaging
2015 Laser Photonics Rev.  article DOI URL 
Abstract: Intravital imaging of large specimens is intrinsically
challenging for postembryonic studies. Selective plane
illumination microscopy (SPIM) has been introduced to
volumetrically visualize organisms used in developmental biology
and experimental genetics. Ideally suited for imaging transparent
samples, SPIM can offer high frame rate imaging with optical
microscopy resolutions and low phototoxicity. However, its
performance quickly deteriorates when applied to opaque tissues.
To overcome this limitation, SPIM optics were merged with optical
and optoacoustic (photoacoustic) readouts. The performance of
this hybrid imaging system was characterized using various
phantoms and by imaging a highly scattering ex vivo juvenile
zebrafish. The results revealed the system's enhanced capability
over that of conventional SPIM for high-resolution imaging over
extended depths of scattering content. The approach described
here may enable future visualization of organisms throughout
their entire development, encompassing regimes in which the
tissue may become opaque.
BibTeX:
@article{Lin2015-ze,
  author = {Lin, Hsiao-Chun Amy and Chekkoury, Andrei and Omar, Murad and
Schmitt-Manderbach, Tobias and Koberstein-Schwarz, Benno and
Mappes, Timo and López-Schier, Hernán and Razansky,
Daniel and Ntziachristos, Vasilis}, title = {Selective plane illumination optical and optoacoustic microscopy
for postembryonic imaging}, journal = {Laser Photonics Rev.}, year = {2015}, url = {http://dx.doi.org/10.1002/lpor.201500120}, doi = {http://dx.doi.org/10.1002/lpor.201500120} }
Liu, J., Bressan, M., Hassel, D., Huisken, J., Staudt, D., Kikuchi, K., Poss, K.D., Mikawa, T. and Stainier, D.Y.R. A dual role for ErbB2 signaling in cardiac trabeculation 2010 Development
Vol. 137(22), pp. 3867-3875 
article DOI URL 
Abstract: Cardiac trabeculation is a crucial morphogenetic process by
which clusters of ventricular cardiomyocytes extrude and
expand into the cardiac jelly to form sheet-like projections.
Although it has been suggested that cardiac trabeculae enhance
cardiac contractility and intra-ventricular conduction, their
exact function in heart development has not been directly
addressed. We found that in zebrafish erbb2 mutants, which we
show completely lack cardiac trabeculae, cardiac function is
significantly compromised, with mutant hearts exhibiting
decreased fractional shortening and an immature conduction
pattern. To begin to elucidate the cellular mechanisms of
ErbB2 function in cardiac trabeculation, we analyzed erbb2
mutant hearts more closely and found that loss of ErbB2
activity resulted in a complete absence of cardiomyocyte
proliferation during trabeculation stages. In addition, based
on data obtained from proliferation, lineage tracing and
transplantation studies, we propose that cardiac trabeculation
is initiated by directional cardiomyocyte migration rather
than oriented cell division, and that ErbB2 cell-autonomously
regulates this process.
BibTeX:
@article{Liu2010-li,
  author = {Liu, Jiandong and Bressan, Michael and Hassel, David and
Huisken, Jan and Staudt, David and Kikuchi, Kazu and Poss,
Kenneth D and Mikawa, Takashi and Stainier, Didier Y R}, title = {A dual role for ErbB2 signaling in cardiac trabeculation}, journal = {Development}, year = {2010}, volume = {137}, number = {22}, pages = {3867--3875}, url = {http://dx.doi.org/10.1242/dev.053736}, doi = {http://dx.doi.org/10.1242/dev.053736} }
Loftus, A.F., Noreng, S., Hsieh, V.L. and Parthasarathy, R. Robust measurement of membrane bending moduli using light
sheet fluorescence imaging of vesicle fluctuations
2013 Langmuir
Vol. 29(47), pp. 14588-14594 
article DOI URL 
Abstract: The mechanical rigidity of lipid membranes is a key
determinant of the energetics of cellular membrane
deformation. Measurements of membrane bending moduli remain
rare, however, and show a large variance, a situation that can
be addressed by the development of improved techniques and by
comparisons between disparate techniques applied to the same
systems. We introduce here the use of selective plane
illumination microscopy (SPIM, also known as light sheet
fluorescence microscopy) to image thermal fluctuations of
giant vesicles. The optical sectioning of SPIM enables
high-speed fluorescence imaging of freely suspended vesicles
and quantification of edge localization precision, yielding
robust fluctuation spectra and rigidity estimates. For both
lipid-only membranes and membranes bound by the intracellular
trafficking protein Sar1p, which lowers membrane rigidity in a
concentration-dependent manner, we show that the resulting
bending modulus values are in close agreement with those
derived from an independent assay based on membrane tether
pulling. We also show that the fluctuation spectra of vesicles
bound by the mammalian Sar1A protein, which stiffens membranes
at high concentrations, are not well fit by a model of
homogeneous quasi-spherical vesicles, suggesting that
SPIM-based analysis can offer insights into spatially
inhomogeneous properties induced by protein assemblies.
BibTeX:
@article{Loftus2013-ts,
  author = {Loftus, Andrew F and Noreng, Sigrid and Hsieh, Vivian L and
Parthasarathy, Raghuveer}, title = {Robust measurement of membrane bending moduli using light
sheet fluorescence imaging of vesicle fluctuations}, journal = {Langmuir}, year = {2013}, volume = {29}, number = {47}, pages = {14588--14594}, url = {http://dx.doi.org/10.1021/la403837d}, doi = {http://dx.doi.org/10.1021/la403837d} }
Lorenzo, C., Frongia, C., Jorand, R., Fehrenbach, J., Weiss, P., Maandhui, A., Gay, G., Ducommun, B. and Lobjois, V. Live cell division dynamics monitoring in 3D large spheroid
tumor models using light sheet microscopy
2011 Cell Div.
Vol. 6, pp. 22 
article DOI URL 
Abstract: BACKGROUND: Multicellular tumor spheroids are models of
increasing interest for cancer and cell biology studies. They
allow considering cellular interactions in exploring cell
cycle and cell division mechanisms. However, 3D imaging of
cell division in living spheroids is technically challenging
and has never been reported. RESULTS: Here, we report a major
breakthrough based on the engineering of multicellular tumor
spheroids expressing an histone H2B fluorescent nuclear
reporter protein, and specifically designed sample holders to
monitor live cell division dynamics in 3D large spheroids
using an home-made selective-plane illumination microscope.
CONCLUSIONS: As illustrated using the antimitotic drug,
paclitaxel, this technological advance paves the way for
studies of the dynamics of cell divion processes in 3D and
more generally for the investigation of tumor cell population
biology in integrated system as the spheroid model.
BibTeX:
@article{Lorenzo2011-nz,
  author = {Lorenzo, Corinne and Frongia, Céline and Jorand,
Raphaël and Fehrenbach, Jérôme and Weiss, Pierre
and Maandhui, Amina and Gay, Guillaume and Ducommun, Bernard
and Lobjois, Valérie}, title = {Live cell division dynamics monitoring in 3D large spheroid
tumor models using light sheet microscopy}, journal = {Cell Div.}, year = {2011}, volume = {6}, pages = {22}, url = {http://dx.doi.org/10.1186/1747-1028-6-22}, doi = {http://dx.doi.org/10.1186/1747-1028-6-22} }
Lucas, M., Kenobi, K., von Wangenheim, D., Vo$, U., Swarup, K., De Smet, I., Van Damme, D., Lawrence, T., Péret, B., Moscardi, E., Barbeau, D., Godin, C., Salt, D., Guyomarc'h, S., Stelzer, E.H.K., Maizel, A., Laplaze, L. and Bennett, M.J. Lateral root morphogenesis is dependent on the mechanical
properties of the overlaying tissues
2013 Proc. Natl. Acad. Sci. U. S. A.
Vol. 110(13), pp. 5229-5234 
article DOI URL 
Abstract: In Arabidopsis, lateral root primordia (LRPs) originate from
pericycle cells located deep within the parental root and have
to emerge through endodermal, cortical, and epidermal tissues.
These overlaying tissues place biomechanical constraints on
the LRPs that are likely to impact their morphogenesis. This
study probes the interplay between the patterns of cell
division, organ shape, and overlaying tissues on LRP
morphogenesis by exploiting recent advances in live plant cell
imaging and image analysis. Our 3D/4D image analysis revealed
that early stage LRPs exhibit tangential divisions that create
a ring of cells corralling a population of rapidly dividing
cells at its center. The patterns of division in the latter
population of cells during LRP morphogenesis are not
stereotypical. In contrast, statistical analysis demonstrated
that the shape of new LRPs is highly conserved. We tested the
relative importance of cell division pattern versus overlaying
tissues on LRP morphogenesis using mutant and transgenic
approaches. The double mutant aurora1 (aur1) aur2 disrupts the
pattern of LRP cell divisions and impacts its growth dynamics,
yet the new organ's dome shape remains normal. In contrast,
manipulating the properties of overlaying tissues disrupted
LRP morphogenesis. We conclude that the interaction with
overlaying tissues, rather than the precise pattern of
divisions, is most important for LRP morphogenesis and
optimizes the process of lateral root emergence.
BibTeX:
@article{Lucas2013-wi,
  author = {Lucas, Mikaël and Kenobi, Kim and von Wangenheim, Daniel
and Vo$, Ute and Swarup, Kamal and De Smet, Ive and Van
Damme, Daniël and Lawrence, Tara and Péret, Benjamin
and Moscardi, Eric and Barbeau, Daniel and Godin, Christophe
and Salt, David and Guyomarc'h, Soazig and Stelzer, Ernst H K
and Maizel, Alexis and Laplaze, Laurent and Bennett, Malcolm J}, title = {Lateral root morphogenesis is dependent on the mechanical
properties of the overlaying tissues}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, year = {2013}, volume = {110}, number = {13}, pages = {5229--5234}, url = {http://dx.doi.org/10.1073/pnas.1210807110}, doi = {http://dx.doi.org/10.1073/pnas.1210807110} }
Luo, X., Salgueiro, Y., Beckerman, S.R., Lemmon, V.P., Tsoulfas, P. and Park, K.K. Three-dimensional evaluation of retinal ganglion cell axon
regeneration and pathfinding in whole mouse tissue after
injury
2013 Exp. Neurol.
Vol. 247, pp. 653-662 
article DOI URL 
Abstract: Injured retinal ganglion cell (RGC) axons do not regenerate
spontaneously, causing loss of vision in glaucoma and after
trauma. Recent studies have identified several strategies that
induce long distance regeneration in the optic nerve. Thus, a
pressing question now is whether regenerating RGC axons can
find their appropriate targets. Traditional methods of
assessing RGC axon regeneration use histological sectioning.
However, tissue sections provide fragmentary information about
axonal trajectory and termination. To unequivocally evaluate
regenerating RGC axons, here we apply tissue clearance and
light sheet fluorescence microscopy (LSFM) to image whole
optic nerve and brain without physical sectioning. In mice
with PTEN/SOCS3 deletion, a condition known to promote robust
regeneration, axon growth followed tortuous paths through the
optic nerve, with many axons reversing course and extending
towards the eye. Such aberrant growth was prevalent in the
proximal region of the optic nerve where strong astroglial
activation is present. In the optic chiasms of PTEN/SOCS3
deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons
project to the opposite optic nerve or to the ipsilateral
optic tract. Following bilateral optic nerve crush, similar
divergent trajectory is seen at the optic chiasm compared to
unilateral crush. Centrally, axonal projection is limited
predominantly to the hypothalamus. Together, we demonstrate
the applicability of LSFM for comprehensive assessment of
optic nerve regeneration, providing in-depth analysis of the
axonal trajectory and pathfinding. Our study indicates
significant axon misguidance in the optic nerve and brain, and
underscores the need for investigation of axon guidance
mechanisms during optic nerve regeneration in adults.
BibTeX:
@article{Luo2013-hj,
  author = {Luo, Xueting and Salgueiro, Yadira and Beckerman, Samuel R and
Lemmon, Vance P and Tsoulfas, Pantelis and Park, Kevin K}, title = {Three-dimensional evaluation of retinal ganglion cell axon
regeneration and pathfinding in whole mouse tissue after
injury}, journal = {Exp. Neurol.}, year = {2013}, volume = {247}, pages = {653--662}, url = {http://dx.doi.org/10.1016/j.expneurol.2013.03.001}, doi = {http://dx.doi.org/10.1016/j.expneurol.2013.03.001} }
Mücksch, J., Spielmann, T., Sisamakis, E. and Widengren, J. Transient state imaging of live cells using single plane
illumination and arbitrary duty cycle excitation pulse trains
2015 J. Biophotonics
Vol. 8(5), pp. 392-400 
article DOI URL 
Abstract: We demonstrate the applicability of Single Plane Illumination
Microscopy to Transient State Imaging (TRAST), offering
sensitive microenvironmental information together with optical
sectioning and reduced overall excitation light exposure of
the specimen. The concept is verified by showing that
transition rates can be determined accurately for free dye in
solution and that fluorophore transition rates can be resolved
pixel-wise in live cells. Furthermore, we derive a new
theoretical framework for analyzing TRAST data acquired with
arbitrary duty cycle pulse trains. By this analysis it is
possible to reduce the overall measurement time and thereby
enhance the frame rates in TRAST imaging.
BibTeX:
@article{Mucksch2015-yp,
  author = {Mücksch, Jonas and Spielmann, Thiemo and Sisamakis,
Evangelos and Widengren, Jerker}, title = {Transient state imaging of live cells using single plane
illumination and arbitrary duty cycle excitation pulse trains}, journal = {J. Biophotonics}, publisher = {Wiley Online Library}, year = {2015}, volume = {8}, number = {5}, pages = {392--400}, url = {http://dx.doi.org/10.1002/jbio.201400015}, doi = {http://dx.doi.org/10.1002/jbio.201400015} }
Müllenbroich, M.C., Silvestri, L., Onofri, L., Costantini, I., Hoff, M.V., Sacconi, L., Iannello, G. and Pavone, F.S. Comprehensive optical and data management infrastructure for
high-throughput light-sheet microscopy of whole mouse brains
2015 Neurophotonics
Vol. 2(4), pp. 041404 
article DOI URL 
Abstract: Comprehensive mapping and quantification of neuronal
projections in the central nervous system requires
high-throughput imaging of large volumes with microscopic
resolution. To this end, we have developed a confocal
light-sheet microscope that has been optimized for
three-dimensional (3-D) imaging of structurally intact
clarified whole-mount mouse brains. We describe the optical
and electromechanical arrangement of the microscope and give
details on the organization of the microscope management
software. The software orchestrates all components of the
microscope, coordinates critical timing and synchronization,
and has been written in a versatile and modular structure
using the LabVIEW language. It can easily be adapted and
integrated to other microscope systems and has been made
freely available to the light-sheet community. The tremendous
amount of data routinely generated by light-sheet microscopy
further requires novel strategies for data handling and
storage. To complete the full imaging pipeline of our
high-throughput microscope, we further elaborate on big data
management from streaming of raw images up to stitching of 3-D
datasets. The mesoscale neuroanatomy imaged at micron-scale
resolution in those datasets allows characterization and
quantification of neuronal projections in unsectioned mouse
brains.
BibTeX:
@article{Mullenbroich2015-gl,
  author = {Müllenbroich, M Caroline and Silvestri, Ludovico and
Onofri, Leonardo and Costantini, Irene and Hoff, Marcel Van't
and Sacconi, Leonardo and Iannello, Giulio and Pavone,
Francesco S}, title = {Comprehensive optical and data management infrastructure for
high-throughput light-sheet microscopy of whole mouse brains}, journal = {Neurophotonics}, publisher = {International Society for Optics and Photonics}, year = {2015}, volume = {2}, number = {4}, pages = {041404}, url = {http://dx.doi.org/10.1117/1.NPh.2.4.041404}, doi = {http://dx.doi.org/10.1117/1.NPh.2.4.041404} }
Mahou, P., Vermot, J., Beaurepaire, E. and Supatto, W. Multicolor two-photon light-sheet microscopy 2014 Nat. Methods
Vol. 11(6), pp. 600-601 
article DOI URL 
BibTeX:
@article{Mahou2014-sf,
  author = {Mahou, Pierre and Vermot, Julien and Beaurepaire, Emmanuel and
Supatto, Willy}, title = {Multicolor two-photon light-sheet microscopy}, journal = {Nat. Methods}, publisher = {Nature Publishing Group}, year = {2014}, volume = {11}, number = {6}, pages = {600--601}, url = {http://dx.doi.org/10.1038/nmeth.2963}, doi = {http://dx.doi.org/10.1038/nmeth.2963} }
Maizel, A., von Wangenheim, D., Federici, F., Haseloff, J. and Stelzer, E.H.K. High-resolution live imaging of plant growth in near
physiological bright conditions using light sheet fluorescence
microscopy
2011 Plant J.
Vol. 68(2), pp. 377-385 
article DOI URL 
Abstract: Most plant growth occurs post-embryonically and is
characterized by the constant and iterative formation of new
organs. Non-invasive time-resolved imaging of intact, fully
functional organisms allows studies of the dynamics involved
in shaping complex organisms. Conventional and confocal
fluorescence microscopy suffer from limitations when whole
living organisms are imaged at single-cell resolution. We
applied light sheet-based fluorescence microscopy to overcome
these limitations and study the dynamics of plant growth. We
designed a special imaging chamber in which the plant is
maintained vertically under controlled illumination with its
leaves in the air and its root in the medium. We show that
minimally invasive, multi-color, three-dimensional imaging of
live Arabidopsis thaliana samples can be achieved at organ,
cellular and subcellular scales over periods of time ranging
from seconds to days with minimal damage to the sample. We
illustrate the capabilities of the method by recording the
growth of primary root tips and lateral root primordia over
several hours. This allowed us to quantify the contribution of
cell elongation to the early morphogenesis of lateral root
primordia and uncover the diurnal growth rhythm of lateral
roots. We demonstrate the applicability of our approach at
varying spatial and temporal scales by following the division
of plant cells as well as the movement of single endosomes in
live growing root samples. This multi-dimensional approach
will have an important impact on plant developmental and cell
biology and paves the way to a truly quantitative description
of growth processes at several scales.
BibTeX:
@article{Maizel2011-wq,
  author = {Maizel, Alexis and von Wangenheim, Daniel and Federici,
Fernán and Haseloff, Jim and Stelzer, Ernst H K}, title = {High-resolution live imaging of plant growth in near
physiological bright conditions using light sheet fluorescence
microscopy}, journal = {Plant J.}, year = {2011}, volume = {68}, number = {2}, pages = {377--385}, url = {http://dx.doi.org/10.1111/j.1365-313X.2011.04692.x}, doi = {http://dx.doi.org/10.1111/j.1365-313X.2011.04692.x} }
Mappes, T., Jahr, N., Csáki, A., Vogler, N., Popp, Jü. and Fritzsche, W. Die Erfindung des Immersions-Ultramikroskops 1912 -- Beginn
der Nanotechnologie?
2012 Angew. Chem. Int. Ed Engl.
Vol. 124(45), pp. 11370-11375 
article DOI URL 
BibTeX:
@article{Mappes2012-jo,
  author = {Mappes, Timo and Jahr, Norbert and Csáki, Andrea and Vogler,
Nadine and Popp, Jürgen and Fritzsche, Wolfgang}, title = {Die Erfindung des Immersions-Ultramikroskops 1912 -- Beginn
der Nanotechnologie?}, journal = {Angew. Chem. Int. Ed Engl.}, publisher = {WILEY-VCH Verlag}, year = {2012}, volume = {124}, number = {45}, pages = {11370--11375}, url = {http://dx.doi.org/10.1002/ange.201204688}, doi = {http://dx.doi.org/10.1002/ange.201204688} }
Martinez-Morales, J.R., Rembold, M., Greger, K., Simpson, J.C., Brown, K.E., Quiring, R., Pepperkok, R., Martin-Bermudo, M.D., Himmelbauer, H. and Wittbrodt, J. ojoplano-mediated basal constriction is essential for optic
cup morphogenesis
2009 Development
Vol. 136(13), pp. 2165-2175 
article DOI URL 
Abstract: Although the vertebrate retina is a well-studied paradigm for
organogenesis, the morphogenetic mechanisms that carve the
architecture of the vertebrate optic cup remain largely
unknown. Understanding how the hemispheric shape of an eye is
formed requires addressing the fundamental problem of how
individual cell behaviour is coordinated to direct epithelial
morphogenesis. Here, we analyze the role of ojoplano (opo), an
uncharacterized gene whose human ortholog is associated with
orofacial clefting syndrome, in the morphogenesis of
epithelial tissues. Most notably, when opo is mutated in
medaka fish, optic cup folding is impaired. We characterize
optic cup morphogenesis in vivo and determine at the cellular
level how opo affects this process. opo encodes a
developmentally regulated transmembrane protein that localizes
to compartments of the secretory pathway and to basal end-feet
of the neuroepithelial precursors. We show that Opo regulates
the polarized localization of focal adhesion components to the
basal cell surface. Furthermore, tissue-specific interference
with integrin-adhesive function impairs optic cup folding,
resembling the ocular phenotype observed in opo mutants. We
propose a model of retinal morphogenesis whereby opo-mediated
formation of focal contacts is required to transmit the
mechanical tensions that drive the macroscopic folding of the
vertebrate optic cup.
BibTeX:
@article{Martinez-Morales2009-dn,
  author = {Martinez-Morales, Juan Ramon and Rembold, Martina and Greger,
Klaus and Simpson, Jeremy C and Brown, Katherine E and
Quiring, Rebecca and Pepperkok, Rainer and Martin-Bermudo,
Maria D and Himmelbauer, Heinz and Wittbrodt, Joachim}, title = {ojoplano-mediated basal constriction is essential for optic
cup morphogenesis}, journal = {Development}, year = {2009}, volume = {136}, number = {13}, pages = {2165--2175}, url = {http://dx.doi.org/10.1242/dev.033563}, doi = {http://dx.doi.org/10.1242/dev.033563} }
Maruyama, A., Oshima, Y., Kajiura-Kobayashi, H., Nonaka, S., Imamura, T. and Naruse, K. Wide field intravital imaging by two-photon-excitation
digital-scanned light-sheet microscopy (2p-DSLM) with a
high-pulse energy laser
2014 Biomed. Opt. Express
Vol. 5(10), pp. 3311-3325 
article DOI URL 
Abstract: Digital-scanned light-sheet microscopy (DSLM) illuminates a
sample in a plane and captures single-photon-excitation
fluorescence images with a camera from a direction
perpendicular to the light sheet. This method is potentially
useful for observing biological specimens, because image
acquisition is relatively fast, resulting in reduction of
phototoxicity. However, DSLM cannot be effectively applied to
high-scattering materials due to the image blur resulting from
thickening of the light sheet by scattered photons. However,
two-photon-excitation DSLM (2p-DSLM) enables collection of
high-contrast image with near infrared (NIR) excitation. In
conventional 2p-DSLM, the minimal excitation volume for
two-photon excitation restricts the field of view. In this
study, we achieved wide-field 2p-DSLM by using a high-pulse
energy fiber laser, and then used this technique to perform
intravital imaging of a small model fish species, medaka
(Oryzias latipes). Wide fields of view (>700 $m) were
achieved by using a low-numerical aperture (NA) objective lens
and high-peak energy NIR excitation at 1040 nm. We also
performed high-speed imaging at near-video rate and
successfully captured the heartbeat movements of a living
medaka fish at 20 frames/sec.
BibTeX:
@article{Maruyama2014-pe,
  author = {Maruyama, Atsushi and Oshima, Yusuke and Kajiura-Kobayashi,
Hiroko and Nonaka, Shigenori and Imamura, Takeshi and Naruse,
Kiyoshi}, title = {Wide field intravital imaging by two-photon-excitation
digital-scanned light-sheet microscopy (2p-DSLM) with a
high-pulse energy laser}, journal = {Biomed. Opt. Express}, year = {2014}, volume = {5}, number = {10}, pages = {3311--3325}, url = {http://dx.doi.org/10.1364/BOE.5.003311}, doi = {http://dx.doi.org/10.1364/BOE.5.003311} }
Matula, P., Kozubek, M., Staier, F. and Hausmann, M. Precise 3D image alignment in micro-axial tomography 2003 J. Microsc.
Vol. 209(Pt 2), pp. 126-142 
article URL 
Abstract: Micro (micro-) axial tomography is a challenging technique in
microscopy which improves quantitative imaging especially in
cytogenetic applications by means of defined sample rotation
under the microscope objective. The advantage of micro-axial
tomography is an effective improvement of the precision of
distance measurements between point-like objects. Under
certain circumstances, the effective (3D) resolution can be
improved by optimized acquisition depending on subsequent,
multi-perspective image recording of the same objects followed
by reconstruction methods. This requires, however, a very
precise alignment of the tilted views. We present a novel
feature-based image alignment method with a precision better
than the full width at half maximum of the point spread
function. The features are the positions (centres of gravity)
of all fluorescent objects observed in the images (e.g. cell
nuclei, fluorescent signals inside cell nuclei, fluorescent
beads, etc.). Thus, real alignment precision depends on the
localization precision of these objects. The method
automatically determines the corresponding objects in
subsequently tilted perspectives using a weighted bipartite
graph. The optimum transformation function is computed in a
least squares manner based on the coordinates of the centres
of gravity of the matched objects. The theoretically feasible
precision of the method was calculated using
computer-generated data and confirmed by tests on real image
series obtained from data sets of 200 nm fluorescent
nano-particles. The advantages of the proposed algorithm are
its speed and accuracy, which means that if enough objects are
included, the real alignment precision is better than the
axial localization precision of a single object. The alignment
precision can be assessed directly from the algorithm's
output. Thus, the method can be applied not only for image
alignment and object matching in tilted view series in order
to reconstruct (3D) images, but also to validate the
experimental performance (e.g. mechanical precision of the
tilting). In practice, the key application of the method is an
improvement of the effective spatial (3D) resolution, because
the well-known spatial anisotropy in light microscopy can be
overcome. This allows more precise distance measurements
between point-like objects.
BibTeX:
@article{Matula2003-nw,
  author = {Matula, P and Kozubek, M and Staier, F and Hausmann, M},
  title = {Precise 3D image alignment in micro-axial tomography},
  journal = {J. Microsc.},
  year = {2003},
  volume = {209},
  number = {Pt 2},
  pages = {126--142},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/12588530}
}
Mayer, Jü., Robert-Moreno, A., Danuser, R., Stein, J.V., Sharpe, J. and Swoger, J. OPTiSPIM: integrating optical projection tomography in light
sheet microscopy extends specimen characterization to
nonfluorescent contrasts
2014 Opt. Lett.
Vol. 39(4), pp. 1053-1056 
article DOI URL 
Abstract: Mesoscopic 3D imaging has become a widely used optical imaging
technique to visualize intact biological specimens. Selective
plane illumination microscopy (SPIM) visualizes samples up to a
centimeter in size with micrometer resolution by 3D data
stitching but is limited to fluorescent contrast. Optical
projection tomography (OPT) works with fluorescent and
nonfluorescent contrasts, but its resolution is limited in large
samples. We present a hybrid setup (OPTiSPIM) combining the
advantages of each technique. The combination of fluorescent and
nonfluorescent high-resolution 3D data into integrated datasets
enables a more extensive representation of mesoscopic biological
samples. The modular concept of the OPTiSPIM facilitates
incorporation of the transmission OPT modality into already
established light sheet based imaging setups.
BibTeX:
@article{Mayer2014-uu,
  author = {Mayer, Jürgen and Robert-Moreno, Alexandre and Danuser,
Renzo and Stein, Jens V and Sharpe, James and Swoger, Jim}, title = {OPTiSPIM: integrating optical projection tomography in light
sheet microscopy extends specimen characterization to
nonfluorescent contrasts}, journal = {Opt. Lett.}, year = {2014}, volume = {39}, number = {4}, pages = {1053--1056}, url = {http://dx.doi.org/10.1364/OL.39.001053}, doi = {http://dx.doi.org/10.1364/OL.39.001053} }
McGorty, R., Liu, H., Kamiyama, D., Dong, Z., Guo, S. and Huang, B. Open-top selective plane illumination microscope for
conventionally mounted specimens
2015 Opt. Express
Vol. 23(12), pp. 16142-16153 
article DOI URL 
Abstract: We have developed a new open-top selective plane illumination
microscope (SPIM) compatible with microfluidic devices,
multi-well plates, and other sample formats used in conventional
inverted microscopy. Its key element is a water prism that
compensates for the aberrations introduced when imaging at 45
degrees through a coverglass. We have demonstrated its unique
high-content imaging capability by recording Drosophila embryo
development in environmentally-controlled microfluidic channels
and imaging zebrafish embryos in 96-well plates. We have also
shown the imaging of C. elegans and moving Drosophila larvae on
coverslips.
BibTeX:
@article{McGorty2015-ht,
  author = {McGorty, Ryan and Liu, Harrison and Kamiyama, Daichi and Dong,
Zhiqiang and Guo, Su and Huang, Bo}, title = {Open-top selective plane illumination microscope for
conventionally mounted specimens}, journal = {Opt. Express}, year = {2015}, volume = {23}, number = {12}, pages = {16142--16153}, url = {http://dx.doi.org/10.1364/OE.23.016142}, doi = {http://dx.doi.org/10.1364/OE.23.016142} }
McLachlan, D. Extreme Focal Depth in Microscopy 1964 Appl. Opt., AO
Vol. 3(9), pp. 1009-1013 
article DOI URL 
Abstract: This paper describes and gives some of the results of using a
microscope which increases the useful depth of observation of an
object in a photograph to many times the focal depth of the lens
system being used. The principle of the microscope is that the
object is illuminated only on the focal plane while the object
is being scanned through that plane. Thus, the out-of-focus
parts of the object are always in darkness, and the final
photographs show high resolution throughout the depth of scan.
The mechanism of scanning is somewhat similar to the
Gregory--Donaldson method, whereas the mechanism of illumination
is similar to that of the Schmaltz slit. This is the first time
that scanning and focal plane illumination have been combined to
attain high resolution at great depths. Of course,
high-frequency scanning would permit direct observation by eye.
BibTeX:
@article{McLachlan1964-pc,
  author = {McLachlan, Dan},
  title = {Extreme Focal Depth in Microscopy},
  journal = {Appl. Opt., AO},
  publisher = {Optical Society of America},
  year = {1964},
  volume = {3},
  number = {9},
  pages = {1009--1013},
  url = {http://www.opticsinfobase.org/ao/abstract.cfm?uri=ao-3-9-1009},
  doi = {http://dx.doi.org/10.1364/AO.3.001009}
}
McLachlan Jr, D. Deep field microscopy 1966 Med. Biol. Illus.
Vol. 16(2), pp. 98-99 
article URL 
BibTeX:
@article{McLachlan1966-ys,
  author = {McLachlan, Jr, D},
  title = {Deep field microscopy},
  journal = {Med. Biol. Illus.},
  year = {1966},
  volume = {16},
  number = {2},
  pages = {98--99},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/5934295}
}
McLachlan Jr., D. The use of deep-field microscopy in crystal morphology 1971 J. Cryst. Growth
Vol. 8(4), pp. 363-371 
article DOI URL 
Abstract: This paper discusses for the first time the limitations of the
deep-field microscope1) from the standpoint of the breadth of
field as well as the usual consideration of depth of field, with
particular consideration to its use in observing the morphology
of grown crystals. Of special consideration in studies of crystal
habit is the use of this instrument for goniometry, that is, the
measurement of interfacial angles by means of contours produced
on the picture of the crystal. The contours are formed by
applying a beam chopper to the optical slit system illuminating
the sample.
BibTeX:
@article{McLachlan1971-er,
  author = {McLachlan, Jr., Dan},
  title = {The use of deep-field microscopy in crystal morphology},
  journal = {J. Cryst. Growth},
  year = {1971},
  volume = {8},
  number = {4},
  pages = {363--371},
  url = {http://www.sciencedirect.com/science/article/pii/0022024871902661},
  doi = {http://dx.doi.org/10.1016/0022-0248(71)90266-1}
}
Mellman, K., Huisken, J., Dinsmore, C., Hoppe, C. and Stainier, D.Y. Fibrillin-2b regulates endocardial morphogenesis in zebrafish 2012 Dev. Biol.
Vol. 372(1), pp. 111-119 
article DOI URL 
Abstract: scotch tape (sco) is a zebrafish cardiac mutant initially
proposed to exhibit a reduced amount of cardiac jelly, the
extracellular matrix between the myocardial and endocardial
layers. We analyzed sco(te382) mutant hearts in detail using
both selective plane illumination microscopy (SPIM) and
transmission electron microscopy (TEM), and observed a
fascinating endocardial defect. Time-lapse SPIM imaging of
wild-type and mutant embryos revealed significant and dynamic
gaps between endocardial cells during development. Although
these gaps close in wild-type animals, they fail to close in
the mutants, ultimately leading to a near complete absence of
endocardial cells in the atrial chamber by the heart looping
stage. TEM analyses confirm the presence of gaps between
endocardial cells in sco mutants, allowing the apparent
leakage of cardiac jelly into the lumen. High-resolution
mapping places the sco(te382) mutation within the fbn2b locus,
which encodes the extracellular matrix protein Fibrillin 2b
(OMIM ID: 121050). Complementation and further phenotypic
analyses confirm that sco is allelic to puff daddy(gw1)
(pfd(gw1)), a null mutant in fbn2b, and that sco(te382) is a
hypomorphic allele of fbn2b. fbn2b belongs to a family of
genes responsible for the assembly of microfibrils throughout
development, and is essential for microfibril structural
integrity. In sco(te382) mutants, Fbn2b is disabled by a
missense mutation in a highly conserved cbEGF domain, which
likely interferes with protein folding. Integrating data
obtained from microscopy and molecular biology, we posit that
this mutation impacts the rigidity of Fbn2b, imparting a
structural defect that weakens endocardial adhesion thereby
resulting in perforated endocardium.
BibTeX:
@article{Mellman2012-dj,
  author = {Mellman, Katharine and Huisken, Jan and Dinsmore, Colin and
Hoppe, Cornelia and Stainier, Didier Y}, title = {Fibrillin-2b regulates endocardial morphogenesis in zebrafish}, journal = {Dev. Biol.}, year = {2012}, volume = {372}, number = {1}, pages = {111--119}, url = {http://dx.doi.org/10.1016/j.ydbio.2012.07.015}, doi = {http://dx.doi.org/10.1016/j.ydbio.2012.07.015} }
Mertz, J. Optical sectioning microscopy with planar or structured
illumination
2011 Nat. Methods
Vol. 8(10), pp. 811-819 
article DOI URL 
Abstract: A key requirement for performing three-dimensional (3D)
imaging using optical microscopes is that they be capable of
optical sectioning by distinguishing in-focus signal from
out-of-focus background. Common techniques for fluorescence
optical sectioning are confocal laser scanning microscopy and
two-photon microscopy. But there is increasing interest in
alternative optical sectioning techniques, particularly for
applications involving high speeds, large fields of view or
long-term imaging. In this Review, I examine two such
techniques, based on planar illumination or structured
illumination. The goal is to describe the advantages and
disadvantages of these techniques.
BibTeX:
@article{Mertz2011-jl,
  author = {Mertz, Jerome},
  title = {Optical sectioning microscopy with planar or structured
illumination}, journal = {Nat. Methods}, publisher = {Nature Publishing Group}, year = {2011}, volume = {8}, number = {10}, pages = {811--819}, url = {http://dx.doi.org/10.1038/nmeth.1709}, doi = {http://dx.doi.org/10.1038/nmeth.1709} }
Mertz, J. and Kim, J. Scanning light-sheet microscopy in the whole mouse brain with
HiLo background rejection
2010 J. Biomed. Opt.
Vol. 15(1), pp. 016027 
article DOI URL 
Abstract: It is well known that light-sheet illumination can enable
optically sectioned wide-field imaging of macroscopic samples.
However, the optical sectioning capacity of a light-sheet
macroscope is undermined by sample-induced scattering or
aberrations that broaden the thickness of the sheet
illumination. We present a technique to enhance the optical
sectioning capacity of a scanning light-sheet microscope by
out-of-focus background rejection. The technique, called HiLo
microscopy, makes use of two images sequentially acquired with
uniform and structured sheet illumination. An optically
sectioned image is then synthesized by fusing high and low
spatial frequency information from both images. The benefits
of combining light-sheet macroscopy and HiLo background
rejection are demonstrated in optically cleared whole mouse
brain samples, using both green fluorescent protein
(GFP)-fluorescence and dark-field scattered light contrast.
BibTeX:
@article{Mertz2010-yq,
  author = {Mertz, Jerome and Kim, Jinhyun},
  title = {Scanning light-sheet microscopy in the whole mouse brain with
HiLo background rejection}, journal = {J. Biomed. Opt.}, year = {2010}, volume = {15}, number = {1}, pages = {016027}, url = {http://dx.doi.org/10.1117/1.3324890}, doi = {http://dx.doi.org/10.1117/1.3324890} }
Mickoleit, M., Schmid, B., Weber, M., Fahrbach, F.O., Hombach, S., Reischauer, S. and Huisken, J. High-resolution reconstruction of the beating zebrafish heart 2014 Nat. Methods
Vol. 11(9), pp. 919-922 
article DOI URL 
Abstract: The heart's continuous motion makes it difficult to capture
high-resolution images of this organ in vivo. We developed
tools based on high-speed selective plane illumination
microscopy (SPIM), offering pristine views into the beating
zebrafish heart. We captured three-dimensional cardiac
dynamics with postacquisition synchronization of multiview
movie stacks, obtained static high-resolution reconstructions
by briefly stopping the heart with optogenetics and resolved
nonperiodic phenomena by high-speed volume scanning with a
liquid lens.
BibTeX:
@article{Mickoleit2014-rs,
  author = {Mickoleit, Michaela and Schmid, Benjamin and Weber, Michael
and Fahrbach, Florian O and Hombach, Sonja and Reischauer,
Sven and Huisken, Jan}, title = {High-resolution reconstruction of the beating zebrafish heart}, journal = {Nat. Methods}, publisher = {Nature Publishing Group}, year = {2014}, volume = {11}, number = {9}, pages = {919--922}, url = {http://dx.doi.org/10.1038/nmeth.3037}, doi = {http://dx.doi.org/10.1038/nmeth.3037} }
Mitchell, T.J., Saunter, C.D., O'Nions, W., Girkin, J.M. and Love, G.D. Adaptive optimisation of illumination beam profiles in
fluorescence microscopy
2015 SPIE BiOS, pp. 93350B-93350B-6  inproceedings DOI URL 
BibTeX:
@inproceedings{Mitchell2015-hz,
  author = {Mitchell, T J and Saunter, C D and O'Nions, W and Girkin, J M
and Love, G D}, title = {Adaptive optimisation of illumination beam profiles in
fluorescence microscopy}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {93350B--93350B--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2203188}, doi = {http://dx.doi.org/10.1117/12.2080310} }
Mohan, K. and Mondal, P.P. MRT letter: Two-photon excitation-based 2pi light-sheet
system for nano-lithography
2015 Microsc. Res. Tech.
Vol. 78(1), pp. 1-7 
article DOI URL 
Abstract: We propose two-photon excitation-based light-sheet technique
for nano-lithography. The system consists of
2$-configured cylindrical lens system with a common
geometrical focus. Upon superposition, the phase-matched
counter-propagating light-sheets result in the generation of
identical and equi spaced nano-bump pattern. Study shows a
feature size of as small as few tens of nanometers with a
inter-bump distance of few hundred nanometers. This technique
overcomes some of the limitations of existing nano-lithography
techniques, thereby, may pave the way for mass-production of
nano-structures. Potential applications can also be found in
optical microscopy, plasmonics, and nano-electronics.
BibTeX:
@article{Mohan2015-pb,
  author = {Mohan, Kavya and Mondal, Partha Pratim},
  title = {MRT letter: Two-photon excitation-based 2pi light-sheet
system for nano-lithography}, journal = {Microsc. Res. Tech.}, year = {2015}, volume = {78}, number = {1}, pages = {1--7}, url = {http://dx.doi.org/10.1002/jemt.22452}, doi = {http://dx.doi.org/10.1002/jemt.22452} }
Mohan, K., Purnapatra, S.B. and Mondal, P.P. Three dimensional fluorescence imaging using multiple
light-sheet microscopy
2014 PLoS One
Vol. 9(6), pp. e96551 
article DOI URL 
Abstract: We developed a multiple light-sheet microscopy (MLSM) system
capable of 3D fluorescence imaging. Employing spatial filter
in the excitation arm of a SPIM system, we successfully
generated multiple light-sheets. This improves upon the
existing SPIM system and is capable of 3D volume imaging by
simultaneously illuminating multiple planes in the sample.
Theta detection geometry is employed for data acquisition from
multiple specimen layers. This detection scheme inherits many
advantages including, background reduction, cross-talk free
fluorescence detection and high-resolution at long working
distance. Using this technique, we generated 5 equi-intense
light-sheets of thickness approximately 7.5 $m with an
inter-sheet separation of 15 $m. Moreover, the
light-sheets generated by MLSM is found to be 2 times thinner
than the state-of-art SPIM system. Imaging of fluorescently
coated yeast cells of size 4 ± 1 $m (encaged in Agarose
gel-matrix) is achieved. Proposed imaging technique may
accelerate the field of fluorescence microscopy, cell biology
and biophotonics.
BibTeX:
@article{Mohan2014-jl,
  author = {Mohan, Kavya and Purnapatra, Subhajit B and Mondal, Partha
Pratim}, title = {Three dimensional fluorescence imaging using multiple
light-sheet microscopy}, journal = {PLoS One}, year = {2014}, volume = {9}, number = {6}, pages = {e96551}, url = {http://dx.doi.org/10.1371/journal.pone.0096551}, doi = {http://dx.doi.org/10.1371/journal.pone.0096551} }
Moosavi, S.H., Gohn-Kreuz, C. and Rohrbach, A. Feedback phase correction of Bessel beams in confocal line
light-sheet microscopy: a simulation study
2013 Appl. Opt.
Vol. 52(23), pp. 5835-5842 
article DOI URL 
Abstract: Confocal line detection has been shown to improve contrast in
light-sheet-based microscopy especially when illuminating the
sample by Bessel beams. Besides their self-reconstructing
capability, the stability in propagation direction of Bessel
beams allows to block the unwanted emission light from the
Bessel beam's ring system. However, due to phase aberrations
induced especially at the border of the specimen, Bessel beams
may not propagate along lines parallel to the slit detector.
Here we present a concept of how to correct the phase of each
incident Bessel beam such that the efficiency of confocal line
detection is improved by up to 200300 The applicability
of the method is verified by the results obtained from
numerical simulations based on the beam propagation method.
BibTeX:
@article{Moosavi2013-ya,
  author = {Moosavi, S Hoda and Gohn-Kreuz, Cristian and Rohrbach,
Alexander}, title = {Feedback phase correction of Bessel beams in confocal line
light-sheet microscopy: a simulation study}, journal = {Appl. Opt.}, year = {2013}, volume = {52}, number = {23}, pages = {5835--5842}, url = {http://dx.doi.org/10.1364/AO.52.005835}, doi = {http://dx.doi.org/10.1364/AO.52.005835} }
Morozov, A.N., Turchin, I.V., Kamenskii, V.A., Fiks, I.I., Lazutkin, A.A., Bezryadkov, D.V., Ivanova, A.A., Toptunov, D.M. and Anokhin, K.V. Fibreoptic fluorescent microscopy in studying biological
objects
2010 Quantum Electron.
Vol. 40(9), pp. 842-846 
article DOI URL 
Abstract: The method of fluorescent microscopy is developed based on
employment of a single-mode fibreoptic channel to provide high
spatial resolution 3D images of large cleared biological
specimens using the 488-nm excitation laser line. The
transverse and axial resolution of the setup is 5 and 13 mm,
respectively. The transversal sample size under investigation
is up to 10 mm. The in-depth scanning range depends on the
sample transparency and reaches 4 mm in the experiment. The 3D
images of whole mouse organs (heart, lungs, brain) and mouse
embryos obtained using autofluorescence or fluorescence of
exogenous markers demonstrate a high contrast and
cellular-level resolution.
BibTeX:
@article{Morozov2010-ql,
  author = {Morozov, A N and Turchin, Il'ya V and Kamenskii, V A and Fiks,
I I and Lazutkin, A A and Bezryadkov, D V and Ivanova, A A and
Toptunov, D M and Anokhin, K V}, title = {Fibreoptic fluorescent microscopy in studying biological
objects}, journal = {Quantum Electron.}, year = {2010}, volume = {40}, number = {9}, pages = {842--846}, url = {http://stacks.iop.org/1063-7818/40/i=9/a=A14?key=crossref.5a3a718582a1049ae0e9a96687e747e5}, doi = {http://dx.doi.org/10.1070/QE2010v040n09ABEH014344} }
Munson, C., Huisken, J., Bit-Avragim, N., Kuo, T., Dong, P.D., Ober, E.A., Verkade, H., Abdelilah-Seyfried, S. and Stainier, D.Y.R. Regulation of neurocoel morphogenesis by Pard6 gamma b 2008 Dev. Biol.
Vol. 324(1), pp. 41-54 
article DOI URL 
Abstract: The Par3/Par6/aPKC protein complex plays a key role in the
establishment and maintenance of apicobasal polarity, a
cellular characteristic essential for tissue and organ
morphogenesis, differentiation and homeostasis. During a
forward genetic screen for liver and pancreas mutants, we
identified a pard6gammab mutant, representing the first known
pard6 mutant in a vertebrate organism. pard6gammab mutants
exhibit defects in epithelial tissue development as well as
multiple lumens in the neural tube. Analyses of the cells
lining the neural tube cavity, or neurocoel, in wildtype and
pard6gammab mutant embryos show that lack of Pard6gammab
function leads to defects in mitotic spindle orientation
during neurulation. We also found that the PB1 (aPKC-binding)
and CRIB (Cdc-42-binding) domains and the KPLG amino acid
sequence within the PDZ domain (Pals1-and Crumbs binding) are
not required for Pard6gammab localization but are essential
for its function in neurocoel morphogenesis. Apical membranes
are reduced, but not completely absent, in mutants lacking the
zygotic, or both the maternal and zygotic, function of
pard6gammab, leading us to examine the localization and
function of the three additional zebrafish Pard6 proteins. We
found that Pard6alpha, but not Pard6beta or Pard6gammaa, could
partially rescue the pard6gammab(s441) mutant phenotypes.
Altogether, these data indicate a previously unappreciated
functional diversity and complexity within the vertebrate
pard6 gene family.
BibTeX:
@article{Munson2008-jy,
  author = {Munson, Chantilly and Huisken, Jan and Bit-Avragim, Nana and
Kuo, Taiyi and Dong, P D and Ober, Elke A and Verkade, Heather
and Abdelilah-Seyfried, Salim and Stainier, Didier Y R}, title = {Regulation of neurocoel morphogenesis by Pard6 gamma b}, journal = {Dev. Biol.}, year = {2008}, volume = {324}, number = {1}, pages = {41--54}, url = {http://dx.doi.org/10.1016/j.ydbio.2008.08.033}, doi = {http://dx.doi.org/10.1016/j.ydbio.2008.08.033} }
Niedworok, C.J., Schwarz, I., Ledderose, J., Giese, Gü., Conzelmann, K.-K. and Schwarz, M.K. Charting monosynaptic connectivity maps by two-color
light-sheet fluorescence microscopy
2012 Cell Rep.
Vol. 2(5), pp. 1375-1386 
article DOI URL 
Abstract: Cellular resolution three-dimensional (3D) visualization of
defined, fluorescently labeled long-range neuronal networks in
the uncut adult mouse brain has been elusive. Here, a
virus-based strategy is described that allowed fluorescent
labeling of centrifugally projecting neuronal populations in
the ventral forebrain and their directly, monosynaptically
connected bulbar interneurons upon a single stereotaxic
injection into select neuronal populations. Implementation of
improved tissue clearing combined with light-sheet
fluorescence microscopy permitted imaging of the resulting
connectivity maps in a single whole-brain scan. Subsequent 3D
reconstructions revealed the exact distribution of the diverse
neuronal ensembles monosynaptically connected with distinct
bulbar interneuron populations. Moreover, rehydratation of
brains after light-sheet fluorescence imaging enabled the
immunohistochemical identification of synaptically connected
neurons. Thus, this study describes a method for identifying
monosynaptic connectivity maps from distinct, virally labeled
neuronal populations that helps in better understanding of
information flow in neural systems.
BibTeX:
@article{Niedworok2012-eo,
  author = {Niedworok, Christian J and Schwarz, Inna and Ledderose, Julia
and Giese, Günter and Conzelmann, Karl-Klaus and
Schwarz, Martin K}, title = {Charting monosynaptic connectivity maps by two-color
light-sheet fluorescence microscopy}, journal = {Cell Rep.}, year = {2012}, volume = {2}, number = {5}, pages = {1375--1386}, url = {http://dx.doi.org/10.1016/j.celrep.2012.10.008}, doi = {http://dx.doi.org/10.1016/j.celrep.2012.10.008} }
Nylk, J., Mitchell, C., Vettenburg, T., Gunn-Moore, F.J. and Dholakia, K. Wavefront shaping of a Bessel light field enhances light sheet
microscopy with scattered light
2014 SPIE BiOS, pp. 89490V-89490V-8  inproceedings DOI URL 
Abstract: abstract Light sheet microscopy has seen a resurgence as it
facilitates rapid, high contrast, volumetric imaging with
minimal sample exposure. Initially developed for imaging
scattered light, this application of light sheet microscopy has
largely been ...
BibTeX:
@inproceedings{Nylk2014-hk,
  author = {Nylk, J and Mitchell, C and Vettenburg, T and Gunn-Moore, F J
and Dholakia, K}, title = {Wavefront shaping of a Bessel light field enhances light sheet
microscopy with scattered light}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {89490V--89490V--8}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1848520}, doi = {http://dx.doi.org/10.1117/12.2038525} }
Olarte, O.E., Andilla, J., Artigas, D. and Loza-Alvarez, P. Decoupled illumination detection in light sheet microscopy for
fast volumetric imaging
2015 Optica
Vol. 2(8), pp. 702 
article DOI URL 
BibTeX:
@article{Olarte2015-eh,
  author = {Olarte, Omar E and Andilla, Jordi and Artigas, David and
Loza-Alvarez, Pablo}, title = {Decoupled illumination detection in light sheet microscopy for
fast volumetric imaging}, journal = {Optica}, year = {2015}, volume = {2}, number = {8}, pages = {702}, url = {https://www.osapublishing.org/abstract.cfm?URI=optica-2-8-702}, doi = {http://dx.doi.org/10.1364/OPTICA.2.000702} }
Olarte, O.E., Licea-Rodriguez, J., Palero, J.A., Gualda, E.J., Artigas, D., Mayer, Jü., Swoger, J., Sharpe, J., Rocha-Mendoza, I., Rangel-Rojo, R. and Loza-Alvarez, P. Image formation by linear and nonlinear digital scanned
light-sheet fluorescence microscopy with Gaussian and Bessel beam
profiles
2012 Biomed. Opt. Express
Vol. 3(7), pp. 1492-1505 
article DOI URL 
Abstract: We present the implementation of a combined digital scanned
light-sheet microscope (DSLM) able to work in the linear and
nonlinear regimes under either Gaussian or Bessel beam excitation
schemes. A complete characterization of the setup is performed
and a comparison of the performance of each DSLM imaging modality
is presented using in vivoCaenorhabditis elegans samples. We
found that the use of Bessel beam nonlinear excitation results in
better image contrast over a wider field of view.
BibTeX:
@article{Olarte2012-om,
  author = {Olarte, Omar E and Licea-Rodriguez, Jacob and Palero, Jonathan A
and Gualda, Emilio J and Artigas, David and Mayer, Jürgen
and Swoger, Jim and Sharpe, James and Rocha-Mendoza, Israel and
Rangel-Rojo, Raul and Loza-Alvarez, Pablo}, title = {Image formation by linear and nonlinear digital scanned
light-sheet fluorescence microscopy with Gaussian and Bessel beam
profiles}, journal = {Biomed. Opt. Express}, year = {2012}, volume = {3}, number = {7}, pages = {1492--1505}, url = {http://dx.doi.org/10.1364/BOE.3.001492}, doi = {http://dx.doi.org/10.1364/BOE.3.001492} }
Opitz, R., Maquet, E., Huisken, J., Antonica, F., Trubiroha, A., Pottier, G., Janssens, V. and Costagliola, S. Transgenic zebrafish illuminate the dynamics of thyroid
morphogenesis and its relationship to cardiovascular
development
2012 Dev. Biol.
Vol. 372(2), pp. 203-216 
article DOI URL 
Abstract: Among the various organs derived from foregut endoderm, the
thyroid gland is unique in that major morphogenic events such
as budding from foregut endoderm, descent into subpharyngeal
mesenchyme and growth expansion occur in close proximity to
cardiovascular tissues. To date, research on thyroid
organogenesis was missing one vital tool-a transgenic model
that allows to track the dynamic changes in thyroid size,
shape and location relative to adjacent cardiovascular tissues
in live embryos. In this study, we generated a novel
transgenic zebrafish line, tg(tg:mCherry), in which robust and
thyroid-specific expression of a membrane version of mCherry
enables live imaging of thyroid development in embryos from
budding stage throughout formation of functional thyroid
follicles. By using various double transgenic models in which
EGFP expression additionally labels cardiovascular structures,
a high coordination was revealed between thyroid organogenesis
and cardiovascular development. Early thyroid development was
found to proceed in intimate contact with the distal
ventricular myocardium and live imaging confirmed that thyroid
budding from the pharyngeal floor is tightly coordinated with
the descent of the heart. Four-dimensional imaging of live
embryos by selective plane illumination microscopy and
3D-reconstruction of confocal images of stained embryos
yielded novel insights into the role of specific pharyngeal
vessels, such as the hypobranchial artery (HA), in guiding
late thyroid expansion along the pharyngeal midline. An
important role of the HA was corroborated by the detailed
examination of thyroid development in various zebrafish models
showing defective cardiovascular development. In combination,
our results from live imaging as well es from
3D-reconstruction of thyroid development in tg(tg:mCherry)
embryos provided a first dynamic view of late thyroid
organogenesis in zebrafish-a critical resource for the design
of future studies addressing the molecular mechanisms of these
thyroid-vasculature interactions.
BibTeX:
@article{Opitz2012-rx,
  author = {Opitz, Robert and Maquet, Emilie and Huisken, Jan and
Antonica, Francesco and Trubiroha, Achim and Pottier,
Gaëlle and Janssens, Véronique and Costagliola,
Sabine}, title = {Transgenic zebrafish illuminate the dynamics of thyroid
morphogenesis and its relationship to cardiovascular
development}, journal = {Dev. Biol.}, year = {2012}, volume = {372}, number = {2}, pages = {203--216}, url = {http://dx.doi.org/10.1016/j.ydbio.2012.09.011}, doi = {http://dx.doi.org/10.1016/j.ydbio.2012.09.011} }
Oshima, Y., Sato, H., Kajiura-Kobayashi, H., Kimura, T., Naruse, K. and Nonaka, S. Light sheet-excited spontaneous Raman imaging of a living fish
by optical sectioning in a wide field Raman microscope
2012 Opt. Express, OE
Vol. 20(15), pp. 16195-16204 
article DOI URL 
Abstract: Spontaneous Raman microscopy is a potentially useful technique
for imaging living cells, tissue and small animals without any
probe or dye labeling. We have developed a spontaneous Raman
imaging system in wide-field view, which we term light
sheet-excited direct Raman spectroscopy (LSDRS). This system,
which we reported previously, consists of a background-free
electrically tunable Ti:Sapphire laser (BF-ETL), a cylindrical
lens, a CCD camera, and a narrow bandpass filter. Here, we have
adapted the LSDRS system for microscopy systems, such as
single-plane illumination microscopy (SPIM) for biomedical
applications, and demonstrated spontaneous Raman imaging of a
living fish. The results suggest that our Raman microscopy
system enables investigation of the differentiation process and
mechanism of iridocytes during development. This is the first
report in which Raman imaging of a living animal was
successfully demonstrated by spontaneous Raman scattering
signals, but not nonlinear Raman effects such as CARS and SRS.
BibTeX:
@article{Oshima2012-pd,
  author = {Oshima, Yusuke and Sato, Hidetoshi and Kajiura-Kobayashi, Hiroko
and Kimura, Tetsuaki and Naruse, Kiyoshi and Nonaka, Shigenori}, title = {Light sheet-excited spontaneous Raman imaging of a living fish
by optical sectioning in a wide field Raman microscope}, journal = {Opt. Express, OE}, publisher = {Optical Society of America}, year = {2012}, volume = {20}, number = {15}, pages = {16195--16204}, url = {http://www.opticsinfobase.org/oe/abstract.cfm?uri=oe-20-15-16195}, doi = {http://dx.doi.org/10.1364/OE.20.016195} }
Ovečka, M., Vaškebová, L., Komis, G., Luptovčiak, I., Smertenko, A. and Šamaj, J. Preparation of plants for developmental and cellular imaging
by light-sheet microscopy
2015 Nat. Protoc.
Vol. 10(8), pp. 1234-1247 
article DOI URL 
Abstract: Long-term fluorescence live-cell imaging experiments have long
been limited by the effects of excitation-induced
phototoxicity. The advent of light-sheet microscopy now allows
users to overcome this limitation by restricting excitation to
a narrow illumination plane. In addition, light-sheet imaging
allows for high-speed image acquisition with uniform
illumination of samples composed of multiple cell layers. The
majority of studies conducted thus far have used custom-built
platforms with specialized hardware and software, along with
specific sample handling approaches. The first versatile
commercially available light-sheet microscope, Lightsheet Z.1,
offers a number of innovative solutions, but it requires
specific strategies for sample handling during long-term
imaging experiments. There are currently no standard
procedures describing the preparation of plant specimens for
imaging with the Lightsheet Z.1. Here we describe a detailed
protocol to prepare plant specimens for light-sheet
microscopy, in which Arabidopsis seeds or seedlings are placed
in solid medium within glass capillaries or fluorinated
ethylene propylene tubes. Preparation of plant material for
imaging may be completed within one working day.
BibTeX:
@article{Ovecka2015-gl,
  author = {Ovečka, Miroslav and Vaškebová, Lenka and Komis,
George and Luptovčiak, Ivan and Smertenko, Andrei and
Šamaj, Jozef}, title = {Preparation of plants for developmental and cellular imaging
by light-sheet microscopy}, journal = {Nat. Protoc.}, year = {2015}, volume = {10}, number = {8}, pages = {1234--1247}, url = {http://dx.doi.org/10.1038/nprot.2015.081}, doi = {http://dx.doi.org/10.1038/nprot.2015.081} }
Pöschinger, T., Renner, A., Eisa, F., Dobosz, M., Strobel, S., Weber, T.G., Brauweiler, R., Kalender, W.A. and Scheuer, W. Dynamic contrast-enhanced micro-computed tomography correlates
with 3-dimensional fluorescence ultramicroscopy in
antiangiogenic therapy of breast cancer xenografts
2014 Invest. Radiol.
Vol. 49(7), pp. 445-456 
article DOI URL 
Abstract: OBJECTIVES: Dynamic contrast-enhanced (DCE) micro-computed
tomography (micro-CT) has emerged as a valuable imaging tool
to noninvasively obtain quantitative physiological biomarkers
of drug effect in preclinical studies of antiangiogenic
compounds. In this study, we explored the ability of DCE
micro-CT to assess the antiangiogenic treatment response in
breast cancer xenografts and correlated the results to the
structural vessel response obtained from 3-dimensional (3D)
fluorescence ultramicroscopy (UM). MATERIAL AND METHODS: Two
groups of tumor-bearing mice (KPL-4) underwent DCE micro-CT
imaging using a fast preclinical dual-source micro-CT system
(TomoScope Synergy Twin, CT Imaging GmbH, Erlangen, Germany).
Mice were treated with either a monoclonal antibody against
the vascular endothelial growth factor or an unspecific
control antibody. Changes in vascular physiology were assessed
measuring the mean value of the relative blood volume (rBV)
and the permeability-surface area product (PS) in different
tumor regions of interest (tumor center, tumor periphery, and
total tumor tissue). Parametric maps of rBV were calculated of
the tumor volume to assess the intratumoral vascular
heterogeneity. Isotropic 3D UM vessel scans were performed
from excised tumor tissue, and automated 3D segmentation
algorithms were used to determine the microvessel density
(MVD), relative vessel volume, and vessel diameters. In
addition, the accumulation of coinjected fluorescence-labeled
trastuzumab was quantified in the UM tissue scans to obtain an
indirect measure of vessel permeability. Results of the DCE
micro-CT were compared with corresponding results obtained by
ex vivo UM. For validation, DCE micro-CT and UM parameters
were compared with conventional histology and tumor volume.
RESULTS: Examination of the parametric rBV maps revealed
significantly different patterns of intratumoral blood supply
between treated and control tumors. Whereas control tumors
showed a characteristic vascular rim pattern with considerably
elevated rBV values in the tumor periphery, treated tumors
showed a widely homogeneous blood supply. Compared with UM,
the physiological rBV maps showed excellent agreement with the
spatial morphology of the intratumoral vascular architecture.
Regional assessment of mean physiological values exhibited a
significant decrease in rBV (P < 0.01) and PS (P < 0.05) in
the tumor periphery after anti-vascular endothelial growth
factor treatment. Structural validation with UM showed a
significant reduction in reduction of relative vessel volume
(rVV) (P < 0.01) and MVD (P < 0.01) in the corresponding tumor
region. The reduction in rBV correlated well with the rVV (R =
0.73 for single values and R = 0.95 for mean values). Spatial
maps of antibody penetration showed a significantly reduced
antibody accumulation (P < 0.01) in the tumor tissue after
treatment and agreed well with the physiological change of PS.
Examination of vessel diameters revealed a size-dependent
antiangiogenic treatment effect, which showed a significant
reduction in MVD (P < 0.001) for vessels with diameters
smaller than 25 $m. No treatment effect was observed by
tumor volume. CONCLUSIONS: Noninvasive DCE micro-CT provides
valuable physiological information of antiangiogenic drug
effect in the intact animal and correlates with ex vivo
structural analysis of 3D UM. The combined use of DCE micro-CT
with UM constitutes a complementary imaging toolset that can
help to enhance our understanding of antiangiogenic drug
mechanisms of action in preclinical drug research.
BibTeX:
@article{Poschinger2014-nt,
  author = {Pöschinger, Thomas and Renner, Anja and Eisa, Fabian and
Dobosz, Michael and Strobel, Steffen and Weber, Thomas G and
Brauweiler, Robert and Kalender, Willi A and Scheuer, Werner}, title = {Dynamic contrast-enhanced micro-computed tomography correlates
with 3-dimensional fluorescence ultramicroscopy in
antiangiogenic therapy of breast cancer xenografts}, journal = {Invest. Radiol.}, year = {2014}, volume = {49}, number = {7}, pages = {445--456}, url = {http://dx.doi.org/10.1097/RLI.0000000000000038}, doi = {http://dx.doi.org/10.1097/RLI.0000000000000038} }
Palayret, M., Armes, H., Basu, S., Watson, A.T., Herbert, A., Lando, D., Etheridge, T.J., Endesfelder, U., Heilemann, M., Laue, E., Carr, A.M., Klenerman, D. and Lee, S.F. Virtual-'light-sheet' single-molecule localisation microscopy
enables quantitative optical sectioning for super-resolution
imaging
2015 PLoS One
Vol. 10(4), pp. e0125438 
article DOI URL 
Abstract: Single-molecule super-resolution microscopy allows imaging of
fluorescently-tagged proteins in live cells with a precision
well below that of the diffraction limit. Here, we demonstrate
3D sectioning with single-molecule super-resolution microscopy
by making use of the fitting information that is usually
discarded to reject fluorophores that emit from above or below
a virtual-'light-sheet', a thin volume centred on the focal
plane of the microscope. We describe an easy-to-use routine
(implemented as an open-source ImageJ plug-in) to quickly
analyse a calibration sample to define and use such a virtual
light-sheet. In addition, the plug-in is easily usable on
almost any existing 2D super-resolution instrumentation. This
optical sectioning of super-resolution images is achieved by
applying well-characterised width and amplitude thresholds to
diffraction-limited spots that can be used to tune the
thickness of the virtual light-sheet. This allows qualitative
and quantitative imaging improvements: by rejecting
out-of-focus fluorophores, the super-resolution image gains
contrast and local features may be revealed; by retaining only
fluorophores close to the focal plane, virtual-'light-sheet'
single-molecule localisation microscopy improves the
probability that all emitting fluorophores will be detected,
fitted and quantitatively evaluated.
BibTeX:
@article{Palayret2015-wn,
  author = {Palayret, Matthieu and Armes, Helen and Basu, Srinjan and
Watson, Adam T and Herbert, Alex and Lando, David and
Etheridge, Thomas J and Endesfelder, Ulrike and Heilemann,
Mike and Laue, Ernest and Carr, Antony M and Klenerman, David
and Lee, Steven F}, title = {Virtual-'light-sheet' single-molecule localisation microscopy
enables quantitative optical sectioning for super-resolution
imaging}, journal = {PLoS One}, year = {2015}, volume = {10}, number = {4}, pages = {e0125438}, url = {http://dx.doi.org/10.1371/journal.pone.0125438}, doi = {http://dx.doi.org/10.1371/journal.pone.0125438} }
Palero, J., Santos, S.I.C.O., Artigas, D. and Loza-Alvarez, P. A simple scanless two-photon fluorescence microscope using
selective plane illumination
2010 Opt. Express
Vol. 18(8), pp. 8491-8498 
article DOI URL 
Abstract: We demonstrate a simple scanless two-photon (2p) excited
fluorescence microscope based on selective plane illumination
microscopy (SPIM). Optical sectioning capability is presented
and depth-resolved imaging of cameleon protein in C. elegans
pharyngeal muscle is implemented.
BibTeX:
@article{Palero2010-hi,
  author = {Palero, Jonathan and Santos, Susana I C O and Artigas, David
and Loza-Alvarez, Pablo}, title = {A simple scanless two-photon fluorescence microscope using
selective plane illumination}, journal = {Opt. Express}, year = {2010}, volume = {18}, number = {8}, pages = {8491--8498}, url = {http://dx.doi.org/10.1364/OE.18.008491}, doi = {http://dx.doi.org/10.1364/OE.18.008491} }
Pampaloni, F., Ansari, N. and Stelzer, E.H.K. High-resolution deep imaging of live cellular spheroids with
light-sheet-based fluorescence microscopy
2013 Cell Tissue Res.
Vol. 352(1), pp. 161-177 
article DOI URL 
Abstract: Conventional two-dimensional cell monolayers do not provide
the geometrical, biochemical and mechanical cues found in real
tissues. Cells in real tissues interact through chemical and
mechanical stimuli with adjacent cells and via the
extracellular matrix. Such a highly interconnected
communication network extends along all three dimensions. This
architecture is lost in two-dimensional cultures. Therefore,
at least in many cases, two-dimensional cell monolayers do not
represent a suitable in vitro tool to characterize accurately
the biology of real tissues. Many studies performed over the
last few years have demonstrated that the differences between
three-dimensional and two-dimensional cultured cells are
striking at the morphological and molecular levels and that
three-dimensional cell cultures can be employed in order to
shrink the gap between real tissues and in vitro cell models.
End-point and long-term imaging of cellular and sub-cellular
processes with fluorescence microscopy provides direct insight
into the physiological behavior of three-dimensional cell
cultures and their response to chemical or mechanical
stimulation. Fluorescence imaging of three-dimensional cell
cultures sets new challenges and imposes specific requirements
concerning the choice of a suitable microscopy technique. Deep
penetration into the specimen, high imaging speed and
ultra-low intensity of the excitation light are key
requirements. Light-sheet-based fluorescence microscopy (LSFM)
offers a favorable combination of these requirements and is
therefore currently established as the technique of choice for
the study of three-dimensional cell cultures. This review
illustrates the benefits of cellular spheroids in the life
sciences and suggests that LSFM is essential for
investigations of cellular and sub-cellular dynamic processes
in three-dimensions over time and space.
BibTeX:
@article{Pampaloni2013-hq,
  author = {Pampaloni, Francesco and Ansari, Nariman and Stelzer, Ernst H
K}, title = {High-resolution deep imaging of live cellular spheroids with
light-sheet-based fluorescence microscopy}, journal = {Cell Tissue Res.}, year = {2013}, volume = {352}, number = {1}, pages = {161--177}, url = {http://dx.doi.org/10.1007/s00441-013-1589-7}, doi = {http://dx.doi.org/10.1007/s00441-013-1589-7} }
Pampaloni, F., Berge, U., Marmaras, A., Horvath, P., Kroschewski, R. and Stelzer, E.H.K. Tissue-culture light sheet fluorescence microscopy (TC-LSFM)
allows long-term imaging of three-dimensional cell cultures
under controlled conditions
2014 Integr. Biol.
Vol. 6(10), pp. 988-998 
article DOI URL 
Abstract: Fluorescence long-term imaging of cellular processes in
three-dimensional cultures requires the control of media supply,
temperature, and pH, as well as minimal photodamage. We describe
a system based on a light sheet fluorescence microscope (LSFM),
which is optimized for long-term, multi-position imaging of
three-dimensional in-gel cell cultures. The system integrates a
stable culture condition control system in the optical path of
the light-sheet microscope. A further essential element is a
biocompatible agarose container suitable for the LSFM, in which
any cell type can be cultured in different gel matrices. The
TC-LSFM allows studying any in vitro cultured cell type reacting
to, dividing in, or migrating through a three-dimensional
extracellular matrix (ECM) gel. For this reason we called it
``tissue culture-LSFM'' (TC-LSFM). The TC-LSFM system allows
fast imaging at multiple locations within a millimeter-sized ECM
gel. This increases the number of analyzed events and allows
testing population effects. As an example, we show the
maturation of a cyst of MDCK (canine kidney epithelial) cells
over a period of three days. Moreover, we imaged, tracked, and
analyzed MDCK cells during the first five days of cell aggregate
formation and discovered a remarkable heterogeneity in cell
cycle lengths and an interesting cell death pattern. Thus,
TC-LSFM allows performing new long-term assays assessing
cellular behavior in three-dimensional ECM-gel cultures. For
example migration, invasion or differentiation in epithelial
cell systems, stem cells, as well as cancer cells can be
investigated.
BibTeX:
@article{Pampaloni2014-eg,
  author = {Pampaloni, Francesco and Berge, Ulrich and Marmaras, Anastasios
and Horvath, Peter and Kroschewski, Ruth and Stelzer, Ernst H K}, title = {Tissue-culture light sheet fluorescence microscopy (TC-LSFM)
allows long-term imaging of three-dimensional cell cultures
under controlled conditions}, journal = {Integr. Biol.}, publisher = {The Royal Society of Chemistry}, year = {2014}, volume = {6}, number = {10}, pages = {988--998}, url = {http://pubs.rsc.org/en/Content/ArticleLanding/2014/IB/C4IB00121D}, doi = {http://dx.doi.org/10.1039/C4IB00121D} }
Pampaloni, F., Chang, B.-J. and Stelzer, E.H.K. Light sheet-based fluorescence microscopy (LSFM) for the
quantitative imaging of cells and tissues
2015 Cell Tissue Res.
Vol. 360(1), pp. 129-141 
article DOI URL 
Abstract: In light sheet-based fluorescence microscopy (LSFM), only the
focal plane is illuminated by a laser light sheet. Hence, only
the fluorophores within a thin volume of the specimen are
excited. This reduces photo-bleaching and photo-toxic effects
by several orders of magnitude compared with any other form of
microscopy. Therefore, LSFM (aka single/selective-plane
illumination microscopy [SPIM] or digitally scanned light
sheet microscopy [DSLM]) is the technique of choice for the
three-dimensional imaging of live or fixed and of small or
large three-dimensional specimens. The parallel recording of
millions of pixels with modern cameras provides an extremely
fast acquisition speed. Recent developments address the
penetration depth, the resolution and the recording speed of
LSFM. The impact of LSFM on research areas such as
three-dimensional cell cultures, neurosciences, plant biology
and developmental biology is increasing at a rapid pace. The
development of high-throughput LSFM is the next leap forward,
allowing the application of LSFM in toxicology and drug
discovery screening.
BibTeX:
@article{Pampaloni2015-lq,
  author = {Pampaloni, Francesco and Chang, Bo-Jui and Stelzer, Ernst H K},
  title = {Light sheet-based fluorescence microscopy (LSFM) for the
quantitative imaging of cells and tissues}, journal = {Cell Tissue Res.}, year = {2015}, volume = {360}, number = {1}, pages = {129--141}, url = {http://dx.doi.org/10.1007/s00441-015-2144-5}, doi = {http://dx.doi.org/10.1007/s00441-015-2144-5} }
Pampaloni, F., Reynaud, E.G. and Stelzer, E.H.K. The third dimension bridges the gap between cell culture and
live tissue
2007 Nat. Rev. Mol. Cell Biol.
Vol. 8(10), pp. 839-845 
article DOI URL 
Abstract: Moving from cell monolayers to three-dimensional (3D) cultures
is motivated by the need to work with cellular models that
mimic the functions of living tissues. Essential cellular
functions that are present in tissues are missed by 'petri
dish'-based cell cultures. This limits their potential to
predict the cellular responses of real organisms. However,
establishing 3D cultures as a mainstream approach requires the
development of standard protocols, new cell lines and
quantitative analysis methods, which include well-suited
three-dimensional imaging techniques. We believe that 3D
cultures will have a strong impact on drug screening and will
also decrease the use of laboratory animals, for example, in
the context of toxicity assays.
BibTeX:
@article{Pampaloni2007-cn,
  author = {Pampaloni, Francesco and Reynaud, Emmanuel G and Stelzer,
Ernst H K}, title = {The third dimension bridges the gap between cell culture and
live tissue}, journal = {Nat. Rev. Mol. Cell Biol.}, year = {2007}, volume = {8}, number = {10}, pages = {839--845}, url = {http://dx.doi.org/10.1038/nrm2236}, doi = {http://dx.doi.org/10.1038/nrm2236} }
Pampaloni, F., Richa, R., Ansari, N. and Stelzer, E.H.K. Live spheroid formation recorded with light sheet-based
fluorescence microscopy
2015 Methods Mol. Biol.
Vol. 1251, pp. 43-57 
article DOI URL 
Abstract: We provide a detailed protocol for a three-dimensional
long-term live imaging of cellular spheroids with light
sheet-based fluorescence microscopy. The protocol allows the
recording of all phases of spheroid formation in three
dimensions, including cell proliferation, aggregation, and
compaction. We employ the human hepatic cell line HepaRG
transfected with the fusion protein H2B-GFP, i.e., a
fluorescing histone. The protocol allows monitoring the effect
of drugs or toxicants.
BibTeX:
@article{Pampaloni2015-et,
  author = {Pampaloni, Francesco and Richa, Roli and Ansari, Nariman and
Stelzer, Ernst H K}, title = {Live spheroid formation recorded with light sheet-based
fluorescence microscopy}, journal = {Methods Mol. Biol.}, publisher = {Springer}, year = {2015}, volume = {1251}, pages = {43--57}, url = {http://dx.doi.org/10.1007/978-1-4939-2080-8_3}, doi = {http://dx.doi.org/10.1007/978-1-4939-2080-8\_3} }
Panier, T., Romano, S.A., Olive, R., Pietri, T., Sumbre, G., Candelier, R. and Debrégeas, G. Fast functional imaging of multiple brain regions in intact
zebrafish larvae using selective plane illumination microscopy
2013 Front. Neural Circuits
Vol. 7, pp. 65 
article DOI URL 
Abstract: The optical transparency and the small dimensions of zebrafish
at the larval stage make it a vertebrate model of choice for
brain-wide in-vivo functional imaging. However, current
point-scanning imaging techniques, such as two-photon or
confocal microscopy, impose a strong limit on acquisition
speed which in turn sets the number of neurons that can be
simultaneously recorded. At 5 Hz, this number is of the order
of one thousand, i.e., approximately 1-2% of the brain. Here
we demonstrate that this limitation can be greatly overcome by
using Selective-plane Illumination Microscopy (SPIM).
Zebrafish larvae expressing the genetically encoded calcium
indicator GCaMP3 were illuminated with a scanned laser sheet
and imaged with a camera whose optical axis was oriented
orthogonally to the illumination plane. This optical
sectioning approach was shown to permit functional imaging of
a very large fraction of the brain volume of 5-9-day-old
larvae with single- or near single-cell resolution. The
spontaneous activity of up to 5,000 neurons was recorded at 20
Hz for 20-60 min. By rapidly scanning the specimen in the
axial direction, the activity of 25,000 individual neurons
from 5 different z-planes (approximately 30% of the entire
brain) could be simultaneously monitored at 4 Hz. Compared to
point-scanning techniques, this imaging strategy thus yields a
20-fold increase in data throughput (number of recorded
neurons times acquisition rate) without compromising the
signal-to-noise ratio (SNR). The extended field of view
offered by the SPIM method allowed us to directly identify
large scale ensembles of neurons, spanning several brain
regions, that displayed correlated activity and were thus
likely to participate in common neural processes. The benefits
and limitations of SPIM for functional imaging in zebrafish as
well as future developments are briefly discussed.
BibTeX:
@article{Panier2013-gy,
  author = {Panier, Thomas and Romano, Sebastián A and Olive,
Raphaël and Pietri, Thomas and Sumbre, Germán and
Candelier, Raphaël and Debrégeas, Georges}, title = {Fast functional imaging of multiple brain regions in intact
zebrafish larvae using selective plane illumination microscopy}, journal = {Front. Neural Circuits}, publisher = {Frontiers Media SA}, year = {2013}, volume = {7}, pages = {65}, url = {http://dx.doi.org/10.3389/fncir.2013.00065}, doi = {http://dx.doi.org/10.3389/fncir.2013.00065} }
Pauli, A., Norris, M.L., Valen, E., Chew, G.-L., Gagnon, J.A., Zimmerman, S., Mitchell, A., Ma, J., Dubrulle, J., Reyon, D., Tsai, S.Q., Joung, J.K., Saghatelian, A. and Schier, A.F. Toddler: an embryonic signal that promotes cell movement via
Apelin receptors
2014 Science
Vol. 343(6172), pp. 1248636 
article DOI URL 
Abstract: It has been assumed that most, if not all, signals regulating
early development have been identified. Contrary to this
expectation, we identified 28 candidate signaling proteins
expressed during zebrafish embryogenesis, including Toddler, a
short, conserved, and secreted peptide. Both absence and
overproduction of Toddler reduce the movement of mesendodermal
cells during zebrafish gastrulation. Local and ubiquitous
production of Toddler promote cell movement, suggesting that
Toddler is neither an attractant nor a repellent but acts
globally as a motogen. Toddler drives internalization of G
protein-coupled APJ/Apelin receptors, and activation of
APJ/Apelin signaling rescues toddler mutants. These results
indicate that Toddler is an activator of APJ/Apelin receptor
signaling, promotes gastrulation movements, and might be the
first in a series of uncharacterized developmental signals.
BibTeX:
@article{Pauli2014-pb,
  author = {Pauli, Andrea and Norris, Megan L and Valen, Eivind and Chew,
Guo-Liang and Gagnon, James A and Zimmerman, Steven and
Mitchell, Andrew and Ma, Jiao and Dubrulle, Julien and Reyon,
Deepak and Tsai, Shengdar Q and Joung, J Keith and
Saghatelian, Alan and Schier, Alexander F}, title = {Toddler: an embryonic signal that promotes cell movement via
Apelin receptors}, journal = {Science}, year = {2014}, volume = {343}, number = {6172}, pages = {1248636}, url = {http://dx.doi.org/10.1126/science.1248636}, doi = {http://dx.doi.org/10.1126/science.1248636} }
Pinto-Teixeira, F., Muzzopappa, M., Swoger, J., Mineo, A., Sharpe, J. and López-Schier, H. Intravital imaging of hair-cell development and regeneration
in the zebrafish
2013 Front. Neuroanat.
Vol. 7, pp. 33 
article DOI URL 
Abstract: Direct videomicroscopic visualization of organ formation and
regeneration in toto is a powerful strategy to study cellular
processes that often cannot be replicated in vitro. Intravital
imaging aims at quantifying changes in tissue architecture or
subcellular organization over time during organ development,
regeneration or degeneration. A general feature of this
approach is its reliance on the optical isolation of defined
cell types in the whole animals by transgenic expression of
fluorescent markers. Here we describe a simple and robust
method to analyze sensory hair-cell development and
regeneration in the zebrafish lateral line by high-resolution
intravital imaging using laser-scanning confocal microscopy
(LSCM) and selective plane illumination microscopy (SPIM). The
main advantage of studying hair-cell regeneration in the
lateral line is that it occurs throughout the life of the
animal, which allows its study in the most natural context. We
detail protocols to achieve continuous videomicroscopy for up
to 68 hours, enabling direct observation of cellular behavior,
which can provide a sensitive assay for the quantitative
classification of cellular phenotypes and cell-lineage
reconstruction. Modifications to this protocol should
facilitate pharmacogenetic assays to identify or validate
otoprotective or reparative drugs for future clinical
strategies aimed at preserving aural function in humans.
BibTeX:
@article{Pinto-Teixeira2013-nr,
  author = {Pinto-Teixeira, Filipe and Muzzopappa, Mariana and Swoger, Jim
and Mineo, Alessandro and Sharpe, James and López-Schier,
Hernán}, title = {Intravital imaging of hair-cell development and regeneration
in the zebrafish}, journal = {Front. Neuroanat.}, year = {2013}, volume = {7}, pages = {33}, url = {http://dx.doi.org/10.3389/fnana.2013.00033}, doi = {http://dx.doi.org/10.3389/fnana.2013.00033} }
Pitrone, P.G., Schindelin, J., Eliceiri, K.W. and Tomancak, P. OpenSPIM: A do-it-yourself open access light sheet fluorescence
microscope
2015 Microscopy and Analysis
Vol. 26(1), pp. 7-11 
article  
BibTeX:
@article{Pitrone2015-rc,
  author = {Pitrone, Peter G and Schindelin, Johannes and Eliceiri, Kevin W
and Tomancak, Pavel}, title = {OpenSPIM: A do-it-yourself open access light sheet fluorescence
microscope}, journal = {Microscopy and Analysis}, year = {2015}, volume = {26}, number = {1}, pages = {7--11} }
Pitrone, P.G., Schindelin, J., Stuyvenberg, L., Preibisch, S., Weber, M., Eliceiri, K.W., Huisken, J. and Tomancak, P. OpenSPIM: an open-access light-sheet microscopy platform 2013 Nat. Methods
Vol. 10(7), pp. 598-599 
article DOI URL 
BibTeX:
@article{Pitrone2013-sy,
  author = {Pitrone, Peter G and Schindelin, Johannes and Stuyvenberg, Luke
and Preibisch, Stephan and Weber, Michael and Eliceiri, Kevin W
and Huisken, Jan and Tomancak, Pavel}, title = {OpenSPIM: an open-access light-sheet microscopy platform}, journal = {Nat. Methods}, year = {2013}, volume = {10}, number = {7}, pages = {598--599}, url = {http://dx.doi.org/10.1038/nmeth.2507}, doi = {http://dx.doi.org/10.1038/nmeth.2507} }
Planchon, T.A., Gao, L., Milkie, D.E., Davidson, M.W., Galbraith, J.A., Galbraith, C.G. and Betzig, E. Rapid three-dimensional isotropic imaging of living cells
using Bessel beam plane illumination
2011 Nat. Methods
Vol. 8(5), pp. 417-423 
article DOI URL 
Abstract: A key challenge when imaging living cells is how to
noninvasively extract the most spatiotemporal information
possible. Unlike popular wide-field and confocal methods,
plane-illumination microscopy limits excitation to the
information-rich vicinity of the focal plane, providing
effective optical sectioning and high speed while minimizing
out-of-focus background and premature photobleaching. Here we
used scanned Bessel beams in conjunction with structured
illumination and/or two-photon excitation to create thinner
light sheets (<0.5 $m) better suited to three-dimensional
(3D) subcellular imaging. As demonstrated by imaging the
dynamics of mitochondria, filopodia, membrane ruffles,
intracellular vesicles and mitotic chromosomes in live cells,
the microscope currently offers 3D isotropic resolution down
to 0.3 $m, speeds up to nearly 200 image planes per
second and the ability to noninvasively acquire hundreds of 3D
data volumes from single living cells encompassing tens of
thousands of image frames.
BibTeX:
@article{Planchon2011-gw,
  author = {Planchon, Thomas A and Gao, Liang and Milkie, Daniel E and
Davidson, Michael W and Galbraith, James A and Galbraith,
Catherine G and Betzig, Eric}, title = {Rapid three-dimensional isotropic imaging of living cells
using Bessel beam plane illumination}, journal = {Nat. Methods}, year = {2011}, volume = {8}, number = {5}, pages = {417--423}, url = {http://dx.doi.org/10.1038/nmeth.1586}, doi = {http://dx.doi.org/10.1038/nmeth.1586} }
Pollmann, C., Hägerling, R. and Kiefer, F. Visualization of lymphatic vessel development, growth, and
function
2014 Adv. Anat. Embryol. Cell Biol.
Vol. 214, pp. 167-186 
article DOI URL 
Abstract: Despite their important physiological and pathophysiological
functions, lymphatic endothelial cells and lymphatic vessels
remain less well studied compared to the blood vascular
system. Lymphatic endothelium differentiates from venous blood
vascular endothelium after initial arteriovenous
differentiation. Only recently by the use of light sheet
microscopy, the precise mechanism of separation of the first
lymphatic endothelial progenitors from the cardinal vein has
been described as delamination followed by mesenchymal cell
migration of lymphatic endothelial cells. Dorsolaterally of
the embryonic cardinal vein, lymphatic endothelial cells
reaggregate to form the first lumenized lymphatic vessels, the
dorsal peripheral longitudinal vessel and the more ventrally
positioned primordial thoracic duct. Despite this progress in
our understanding of the first lymph vessel formation,
intravital observation of lymphatic vessel behavior in the
intact organism, during development and in the adult, is
prerequisite to a precise understanding of this tissue.
Transgenic models and two-photon microscopy, in combination
with optical windows, have made live intravital imaging
possible: however, new imaging modalities and novel approaches
promise gentler, more physiological, and longer intravital
imaging of lymphatic vessels.
BibTeX:
@article{Pollmann2014-sf,
  author = {Pollmann, Cathrin and Hägerling, René and Kiefer,
Friedemann}, title = {Visualization of lymphatic vessel development, growth, and
function}, journal = {Adv. Anat. Embryol. Cell Biol.}, year = {2014}, volume = {214}, pages = {167--186}, url = {http://dx.doi.org/10.1007/978-3-7091-1646-3_13}, doi = {http://dx.doi.org/10.1007/978-3-7091-1646-3\_13} }
Preibisch, S., Amat, F., Stamataki, E., Sarov, M., Singer, R.H., Myers, E. and Tomancak, P. Efficient Bayesian-based multiview deconvolution 2014 Nat. Methods
Vol. 11(6), pp. 645-648 
article DOI URL 
Abstract: Light-sheet fluorescence microscopy is able to image large
specimens with high resolution by capturing the samples from
multiple angles. Multiview deconvolution can substantially
improve the resolution and contrast of the images, but its
application has been limited owing to the large size of the
data sets. Here we present a Bayesian-based derivation of
multiview deconvolution that drastically improves the
convergence time, and we provide a fast implementation using
graphics hardware.
BibTeX:
@article{Preibisch2014-fc,
  author = {Preibisch, Stephan and Amat, Fernando and Stamataki, Evangelia
and Sarov, Mihail and Singer, Robert H and Myers, Eugene and
Tomancak, Pavel}, title = {Efficient Bayesian-based multiview deconvolution}, journal = {Nat. Methods}, year = {2014}, volume = {11}, number = {6}, pages = {645--648}, url = {http://dx.doi.org/10.1038/nmeth.2929}, doi = {http://dx.doi.org/10.1038/nmeth.2929} }
Preibisch, S., Ejsmont, R., Rohlfing, T. and Tomancak, R. Towards digital representation of Drosophila embryogenesis 2008 Biomedical Imaging: From Nano to Macro, 2008. ISBI 2008. 5th
IEEE International Symposium on, pp. 324-327 
inproceedings DOI URL 
Abstract: Animal development can be described as a complex, three-
dimensional cellular system that changes dramatically across
time as a consequence of cell proliferation, differentiation and
movement. Using Drosophila embryogenesis as a model we are
developing molecular, imaging and image analysis techniques to
record an entire developmental system at cellular resolution. We
image Drosophila embryos expressing fluorescent markers in toto
using single plane illumination microscopy (SPIM). SPIM offers
the unique ability to image large living biological specimens in
their entirety by acquiring image stacks from multiple angles
while also providing high temporal resolution necessary for
following dynamic developmental processes. We have developed an
image analysis pipeline that efficiently processes long-term
time-lapse multi-view SPIM data by aligning the different angles
with high precision for a single time point and propagating the
alignment parameters throughout the time series. The registered
views are fused using an approach that evaluates the image
content in each view.
BibTeX:
@inproceedings{Preibisch2008-gh,
  author = {Preibisch, S and Ejsmont, R and Rohlfing, T and Tomancak, R},
  title = {Towards digital representation of Drosophila embryogenesis},
  booktitle = {Biomedical Imaging: From Nano to Macro, 2008. ISBI 2008. 5th
IEEE International Symposium on}, year = {2008}, pages = {324--327}, url = {http://dx.doi.org/10.1109/ISBI.2008.4540998}, doi = {http://dx.doi.org/10.1109/ISBI.2008.4540998} }
Preibisch, S., Rohlfing, T., Hasak, M.P. and Tomancak, P. Mosaicing of single plane illumination microscopy images using
groupwise registration and fast content-based image fusion
2008
Vol. 6914Medical Imaging, pp. 69140E-69140E-8 
inproceedings DOI URL 
Abstract: Single Plane Illumination Microscopy (SPIM; Huisken et al.,
Nature 305(5686):1007-1009, 2004) is an emerging microscopic
technique that enables live imaging of large biological
specimens in their entirety. By imaging the living biological
sample from multiple angles SPIM has the potential to achieve
isotropic resolution throughout even relatively large biological
specimens. For every angle, however, only a relatively shallow
section of the specimen is imaged with high resolution, whereas
deeper regions appear increasingly blurred. In order to produce
a single, uniformly high resolution image, we propose here an
image mosaicing algorithm that combines state of the art
groupwise image registration for alignment with content-based
image fusion to prevent degrading of the fused image due to
regional blurring of the input images. For the registration
stage, we introduce an application-specific groupwise
transformation model that incorporates per-image as well as
groupwise transformation parameters. We also propose a new
fusion algorithm based on Gaussian filters, which is
substantially faster than fusion based on local image entropy.
We demonstrate the performance of our mosaicing method on data
acquired from living embryos of the fruit fly, Drosophila ,
using four and eight angle acquisitions.
BibTeX:
@inproceedings{Preibisch2008-st,
  author = {Preibisch, Stephan and Rohlfing, Torsten and Hasak, Michael P
and Tomancak, Pavel}, title = {Mosaicing of single plane illumination microscopy images using
groupwise registration and fast content-based image fusion}, booktitle = {Medical Imaging}, publisher = {International Society for Optics and Photonics}, year = {2008}, volume = {6914}, pages = {69140E--69140E--8}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=827745}, doi = {http://dx.doi.org/10.1117/12.770893} }
Preibisch, S., Saalfeld, S., Rohlfing, T. and Tomancak, P. Bead-based mosaicing of single plane illumination microscopy
images using geometric local descriptor matching
2009
Vol. 7259SPIE Medical Imaging, pp. 72592S-72592S-10 
inproceedings DOI URL 
BibTeX:
@inproceedings{Preibisch2009-bc,
  author = {Preibisch, Stephan and Saalfeld, Stephan and Rohlfing, Torsten
and Tomancak, Pavel}, title = {Bead-based mosaicing of single plane illumination microscopy
images using geometric local descriptor matching}, booktitle = {SPIE Medical Imaging}, publisher = {International Society for Optics and Photonics}, year = {2009}, volume = {7259}, pages = {72592S--72592S--10}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1335480}, doi = {http://dx.doi.org/10.1117/12.812612} }
Preibisch, S., Saalfeld, S., Schindelin, J. and Tomancak, P. Software for bead-based registration of selective plane
illumination microscopy data
2010 Nat. Methods
Vol. 7(6), pp. 418-419 
article DOI URL 
Abstract: Selective plane illumination microscopy (SPIM) 1 allows
isotropic, time-lapse, in toto imaging of large, living
biological specimens by acquiring three-dimensional (3D) images
of the same sample from multiple angles (views). However, to
realize the potential of multiview SPIM ...
BibTeX:
@article{Preibisch2010-st,
  author = {Preibisch, Stephan and Saalfeld, Stephan and Schindelin, Johannes
and Tomancak, Pavel}, title = {Software for bead-based registration of selective plane
illumination microscopy data}, journal = {Nat. Methods}, year = {2010}, volume = {7}, number = {6}, pages = {418--419}, url = {http://dx.doi.org/10.1038/nmeth0610-418}, doi = {http://dx.doi.org/10.1038/nmeth0610-418} }
Psycharakis, S., Zacharopoulos, A., Ripoll, J., Zacharakis, G., Riekher, M. and Tavernarakis, N. Optical projection tomography and light sheet microscopy for
imaging in biological specimens a comparison study
2014 Imaging Systems and Techniques (IST), 2014 IEEE
International Conference on, pp. 211-215 
inproceedings DOI URL 
Abstract: Optical Projection Tomography and Light Sheet Microscopy have
been recent revolutionary techniques for imaging in biological
specimens with 3D volume rendering of high resolution. A
comparison study is performed on the basic aspects of each
modality and combined application on selected specimen is
presented. The imaging performance characteristics of the two
modalities are calculated and analyzed. Studies in model
organisms such as Caenorhabditis Elegans and Parhyale, are shown
and the significance of each method is discussed in relation to
the specific applications they are best tailored for.
BibTeX:
@inproceedings{Psycharakis2014-yd,
  author = {Psycharakis, S and Zacharopoulos, A and Ripoll, J and
Zacharakis, G and Riekher, M and Tavernarakis, N}, title = {Optical projection tomography and light sheet microscopy for
imaging in biological specimens a comparison study}, booktitle = {Imaging Systems and Techniques (IST), 2014 IEEE
International Conference on}, year = {2014}, pages = {211--215}, url = {http://dx.doi.org/10.1109/IST.2014.6958475}, doi = {http://dx.doi.org/10.1109/IST.2014.6958475} }
Purnapatra, S.B., Mohan, K. and Mondal, P.P. Generation of multiple sheets of light using spatial-filtering
technique
2014 Opt. Lett.
Vol. 39(16), pp. 4715-4718 
article DOI URL 
Abstract: We develop an optical system for generating multiple light
sheets. This is enabled by employing a special class of spatial
filters in a cylindrical lens geometry. The proposed binary
filter placed at the back aperture of the cylindrical lens
results in the generation of a periodic transverse pattern
extending along the z axis (i.e., multiple light sheets).
Experimental results confirm the generation of multiple light
sheets of thickness 6.6 $m with an intersheet spacing of 13.4
$m. The proposed imaging technique may facilitate
three-dimensional imaging in nano-optics, fluorescence
microscopy, and nanobiology.
BibTeX:
@article{Purnapatra2014-hm,
  author = {Purnapatra, Subhajit B and Mohan, Kavya and Mondal, Partha P},
  title = {Generation of multiple sheets of light using spatial-filtering
technique}, journal = {Opt. Lett.}, year = {2014}, volume = {39}, number = {16}, pages = {4715--4718}, url = {http://dx.doi.org/10.1364/OL.39.004715}, doi = {http://dx.doi.org/10.1364/OL.39.004715} }
Purnapatra, S.B. and Mondal, P.P. Generation of extended light-sheets for single and multi-photon
fluorescence microscopy
2013 Appl. Phys. Lett.
Vol. 103(4), pp. 043701 
article DOI URL 
Abstract: We theoretically propose and computationally demonstrate the
generation of extended light-sheet for fluorescence microscopy.
This is made possible by the introduction of a specially
designed double-window spatial filter that allows the light to
pass through the periphery and center of a cylindrical lens.
When illuminated with a plane wave, the proposed filter results
in an extended depth-of-focus along with side-lobes which are
due to other interferences in the transverse focal plane.
Computational studies show a maximum extension of light-sheet by
3.38 times for single photon excitation and 3.68 times for
multiphoton excitation as compared to state-of-art single plane
illumination microscopy system. This technique may facilitate
the study of large biological specimens (such as Zebrafish
embryo and tissue) with high spatial resolution and reduced
photobleaching.
BibTeX:
@article{Purnapatra2013-re,
  author = {Purnapatra, Subhajit B and Mondal, Partha Pratim},
  title = {Generation of extended light-sheets for single and multi-photon
fluorescence microscopy}, journal = {Appl. Phys. Lett.}, publisher = {AIP Publishing}, year = {2013}, volume = {103}, number = {4}, pages = {043701}, url = {http://scitation.aip.org/content/aip/journal/apl/103/4/10.1063/1.4816419}, doi = {http://dx.doi.org/10.1063/1.4816419} }
Rühland, S., Wechselberger, A., Spitzweg, C., Huss, R., Nelson, P.J. and Harz, H. Quantification of in vitro mesenchymal stem cell invasion into
tumor spheroids using selective plane illumination microscopy
2015 J. Biomed. Opt.
Vol. 20(4), pp. 040501 
article DOI URL 
Abstract: Mesenchymal stem cell (MSC) homing and integration into tumors
are under evaluation for clinical application. This approach
requires the identification of conditions for optimal tumor
invasion. We describe a tool for the in vitro comparison of
parameters influencing invasion. Human MSC added to
experimental tumor spheroids variably migrates toward the
center of the structure. To determine MSC distribution inside
the three-dimensional specimen, spatial analysis was performed
using selective plane illumination microscopy. A standardized
method to quantify and compare the invasion potential of
variably treated MSC into experimental tumor environments
allows efficient screening for optimizing conditions.
BibTeX:
@article{Ruhland2015-cu,
  author = {Rühland, Svenja and Wechselberger, Alexandra and
Spitzweg, Christine and Huss, Ralf and Nelson, Peter J and
Harz, Hartmann}, title = {Quantification of in vitro mesenchymal stem cell invasion into
tumor spheroids using selective plane illumination microscopy}, journal = {J. Biomed. Opt.}, year = {2015}, volume = {20}, number = {4}, pages = {040501}, url = {http://dx.doi.org/10.1117/1.JBO.20.4.040501}, doi = {http://dx.doi.org/10.1117/1.JBO.20.4.040501} }
Razansky, D., Vinegoni, C. and Ntziachristos, V. Imaging of mesoscopic-scale organisms using selective-plane
optoacoustic tomography
2009 Phys. Med. Biol.
Vol. 54(9), pp. 2769-2777 
article DOI URL 
Abstract: Mesoscopic-scale living organisms (i.e. 1 mm to 1 cm sized)
remain largely inaccessible by current optical imaging methods
due to intensive light scattering in tissues. Therefore,
imaging of many important model organisms, such as insects,
fishes, worms and similarly sized biological specimens, is
currently limited to embryonic or other transparent stages of
development. This makes it difficult to relate embryonic
cellular and molecular mechanisms to consequences in organ
function and animal behavior in more advanced stages and
adults. Herein, we have developed a selective-plane
illumination optoacoustic tomography technique for in vivo
imaging of optically diffusive organisms and tissues. The
method is capable of whole-body imaging at depths from the
sub-millimeter up to centimeter range with a scalable spatial
resolution in the order of magnitude of a few tenths of
microns. In contrast to pure optical methods, the spatial
resolution here is not determined nor limited by light
diffusion; therefore, such performance cannot be achieved by
any other optical imaging technology developed so far. The
utility of the method is demonstrated on several whole-body
models and small-animal extremities.
BibTeX:
@article{Razansky2009-td,
  author = {Razansky, Daniel and Vinegoni, Claudio and Ntziachristos,
Vasilis}, title = {Imaging of mesoscopic-scale organisms using selective-plane
optoacoustic tomography}, journal = {Phys. Med. Biol.}, year = {2009}, volume = {54}, number = {9}, pages = {2769--2777}, url = {http://dx.doi.org/10.1088/0031-9155/54/9/012}, doi = {http://dx.doi.org/10.1088/0031-9155/54/9/012} }
Regmi, R., Mohan, K. and Mondal, P.P. High resolution light-sheet based high-throughput imaging
cytometry system enables visualization of intra-cellular
organelles
2014 AIP Adv.
Vol. 4(9), pp. 097125 
article DOI URL 
Abstract: Visualization of intracellular organelles is achieved using a
newly developed high throughput imaging cytometry system. This
system interrogates the microfluidic channel using a sheet of
light rather than the existing point-based scanning techniques.
The advantages of the developed system are many, including,
single-shot scanning of specimens flowing through the
microfluidic channel at flow rate ranging from micro- to nano-
lit./min. Moreover, this opens-up in-vivo imaging of
sub-cellular structures and simultaneous cell counting in an
imaging cytometry system. We recorded a maximum count of 2400
cells/min at a flow-rate of 700 nl/min, and simultaneous
visualization of fluorescently-labeled mitochondrial network in
HeLa cells during flow. The developed imaging cytometry system
may find immediate application in biotechnology, fluorescence
microscopy and nano-medicine.
BibTeX:
@article{Regmi2014-hf,
  author = {Regmi, Raju and Mohan, Kavya and Mondal, Partha Pratim},
  title = {High resolution light-sheet based high-throughput imaging
cytometry system enables visualization of intra-cellular
organelles}, journal = {AIP Adv.}, publisher = {AIP Publishing}, year = {2014}, volume = {4}, number = {9}, pages = {097125}, url = {http://scitation.aip.org/content/aip/journal/adva/4/9/10.1063/1.4896260}, doi = {http://dx.doi.org/10.1063/1.4896260} }
Regmi, R., Mohan, K. and Mondal, P.P. MRT letter: light sheet based imaging flow cytometry on a
microfluidic platform
2013 Microsc. Res. Tech.
Vol. 76(11), pp. 1101-1107 
article DOI URL 
Abstract: We propose a light sheet based imaging flow cytometry
technique for simultaneous counting and imaging of cells on a
microfluidic platform. Light sheet covers the entire
microfluidic channel and thus omits the necessity of flow
focusing and point scanning based technology. Another
advantage lies in the orthogonal detection geometry that
totally cuts-off the incident light, thereby substantially
reducing the background in the detection. Compared to the
existing state-of-art techniques the proposed technique shows
marked improvement. Using fluorescently-coated Saccharomyces
cerevisiae cells we have recorded cell counting with
throughput as high as 2,090 cells/min in the low flow rate
regime and were able to image the individual cells on-the-go.
Overall, the proposed system is cost-effective and simple in
channel geometry with the advantage of efficient counting in
operational regime of low laminar flow. This technique may
advance the emerging field of microfluidic based cytometry for
applications in nanomedicine and point of care diagnostics.
BibTeX:
@article{Regmi2013-td,
  author = {Regmi, Raju and Mohan, Kavya and Mondal, Partha P},
  title = {MRT letter: light sheet based imaging flow cytometry on a
microfluidic platform}, journal = {Microsc. Res. Tech.}, year = {2013}, volume = {76}, number = {11}, pages = {1101--1107}, url = {http://dx.doi.org/10.1002/jemt.22296}, doi = {http://dx.doi.org/10.1002/jemt.22296} }
Reissig, J. Ultramikroskopische Beobachtungen 1908 Ann. Phys.
Vol. 332(11), pp. 186-212 
article DOI URL 
BibTeX:
@article{Reissig1908-yo,
  author = {Reissig, J},
  title = {Ultramikroskopische Beobachtungen},
  journal = {Ann. Phys.},
  publisher = {WILEY-VCH Verlag},
  year = {1908},
  volume = {332},
  number = {11},
  pages = {186--212},
  url = {http://dx.doi.org/10.1002/andp.19083321110},
  doi = {http://dx.doi.org/10.1002/andp.19083321110}
}
Renaud, O., Heintzmann, R., Sáez-Cirión, A. and Schnelle, T. A system and methodology for high-content visual screening of
individual intact living cells in łdots
2007 Proc. SPIE  article URL 
Abstract: - visual screening of individual intact living cells in
suspension. [Proceedings of SPIE 6441, 64410Q (2007)].
BibTeX:
@article{Renaud2007-ym,
  author = {Renaud, O and Heintzmann, Rainer and Sáez-Cirión, A and
Schnelle, T}, title = {A system and methodology for high-content visual screening of
individual intact living cells in łdots}, journal = {Proc. SPIE}, year = {2007}, url = {http://link.aip.org/link/?PSISDG/6441/64410Q/1} }
Renaud, O., Vi na, J., Yu, Y., Machu, C., Trouvé, A., Van der Voort, H., Chalmond, B. and Shorte, S.L. High-resolution 3-D imaging of living cells in suspension
using confocal axial tomography
2008 Biotechnol. J.
Vol. 3(1), pp. 53-62 
article DOI URL 
Abstract: Conventional flow cytometry (FC) methods report optical
signals integrated from individual cells at throughput rates
as high as thousands of cells per second. This is further
combined with the powerful utility to subsequently sort and/or
recover the cells of interest. However, these methods cannot
extract spatial information. This limitation has prompted
efforts by some commercial manufacturers to produce
state-of-the-art commercial flow cytometry systems allowing
fluorescence images to be recorded by an imaging detector.
Nonetheless, there remains an immediate and growing need for
technologies facilitating spatial analysis of fluorescent
signals from cells maintained in flow suspension. Here, we
report a novel methodological approach to this problem that
combines micro-fluidic flow, and microelectrode
dielectric-field control to manipulate, immobilize and image
individual cells in suspension. The method also offers unique
possibilities for imaging studies on cells in suspension. In
particular, we report the system's immediate utility for
confocal ``axial tomography'' using micro-rotation imaging and
show that it greatly enhances 3-D optical resolution compared
with conventional light reconstruction (deconvolution) image
data treatment. That the method we present here is relatively
rapid and lends itself to full automation suggests its
eventual utility for 3-D imaging cytometry.
BibTeX:
@article{Renaud2008-ck,
  author = {Renaud, Olivier and Viña, Jose and Yu, Yong and Machu,
Christophe and Trouvé, Alain and Van der Voort, Hans and
Chalmond, Bernard and Shorte, Spencer L}, title = {High-resolution 3-D imaging of living cells in suspension
using confocal axial tomography}, journal = {Biotechnol. J.}, year = {2008}, volume = {3}, number = {1}, pages = {53--62}, url = {http://dx.doi.org/10.1002/biot.200700188}, doi = {http://dx.doi.org/10.1002/biot.200700188} }
Reynaud, E.G., Krzic, U., Greger, K. and Stelzer, E.H.K. Light sheet-based fluorescence microscopy: more dimensions,
more photons, and less photodamage
2008 HFSP J.
Vol. 2(5), pp. 266-275 
article DOI URL 
Abstract: Light-sheet-based fluorescence microscopy (LSFM) is a
fluorescence technique that combines optical sectioning, the
key capability of confocal and two-photon fluorescence
microscopes with multiple-view imaging, which is used in
optical tomography. In contrast to conventional wide-field and
confocal fluorescence microscopes, a light sheet illuminates
only the focal plane of the detection objective lens from the
side. Excitation is, thus, restricted to the fluorophores in
the volume near the focal plane. This provides optical
sectioning and allows the use of regular cameras in the
detection process. Compared to confocal fluorescence
microscopy, LSFM reduces photo bleaching and photo toxicity by
up to three orders of magnitude. In LSFM, the specimen is
embedded in a transparent block of hydrogel and positioned
relative to the stationary light sheet using precise motorized
translation and rotation stages. This feature is used to image
any plane in a specimen. Additionally, multiple views obtained
along different angles can be combined into a single data set
with an improved resolution. LSFMs are very well suited for
imaging large live specimens over long periods of time.
However, they also perform well with very small specimens such
as single yeast cells. This perspective introduces the
principles of LSFM, explains the challenges of specimen
preparation, and introduces the basics of a microscopy that
takes advantage of multiple views.
BibTeX:
@article{Reynaud2008-wf,
  author = {Reynaud, Emmanuel G and Krzic, Uros and Greger, Klaus and
Stelzer, Ernst H K}, title = {Light sheet-based fluorescence microscopy: more dimensions,
more photons, and less photodamage}, journal = {HFSP J.}, year = {2008}, volume = {2}, number = {5}, pages = {266--275}, url = {http://dx.doi.org/10.2976/1.2974980}, doi = {http://dx.doi.org/10.2976/1.2974980} }
Reynaud, E.G., Peychl, J., Huisken, J. and Tomancak, P. Guide to light-sheet microscopy for adventurous biologists 2015 Nat. Methods
Vol. 12(1), pp. 30-34 
article DOI URL 
BibTeX:
@article{Reynaud2015-zg,
  author = {Reynaud, Emmanuel G and Peychl, Jan and Huisken, Jan and
Tomancak, Pavel}, title = {Guide to light-sheet microscopy for adventurous biologists}, journal = {Nat. Methods}, year = {2015}, volume = {12}, number = {1}, pages = {30--34}, url = {http://dx.doi.org/10.1038/nmeth.3222}, doi = {http://dx.doi.org/10.1038/nmeth.3222} }
Reynaud, E.G. and Tomancak, P. Meeting report: first light sheet based fluorescence
microscopy workshop
2010 Biotechnol. J.
Vol. 5(8), pp. 798-804 
article DOI URL 
BibTeX:
@article{Reynaud2010-xj,
  author = {Reynaud, Emmanuel G and Tomancak, Pavel},
  title = {Meeting report: first light sheet based fluorescence
microscopy workshop}, journal = {Biotechnol. J.}, year = {2010}, volume = {5}, number = {8}, pages = {798--804}, url = {http://dx.doi.org/10.1002/biot.201000177}, doi = {http://dx.doi.org/10.1002/biot.201000177} }
Rieckher, M., Kyparissidis-Kokkinidis, I., Zacharopoulos, A., Kourmoulakis, G., Tavernarakis, N., Ripoll, J. and Zacharakis, G. A customized light sheet microscope to measure spatio-temporal
protein dynamics in small model organisms
2015 PLoS One
Vol. 10(5), pp. e0127869 
article DOI URL 
Abstract: We describe a customizable and cost-effective light sheet
microscopy (LSM) platform for rapid three-dimensional imaging
of protein dynamics in small model organisms. The system is
designed for high acquisition speeds and enables extended
time-lapse in vivo experiments when using fluorescently
labeled specimens. We demonstrate the capability of the setup
to monitor gene expression and protein localization during
ageing and upon starvation stress in longitudinal studies in
individual or small groups of adult Caenorhabditis elegans
nematodes. The system is equipped to readily perform
fluorescence recovery after photobleaching (FRAP), which
allows monitoring protein recovery and distribution under low
photobleaching conditions. Our imaging platform is designed to
easily switch between light sheet microscopy and optical
projection tomography (OPT) modalities. The setup permits
monitoring of spatio-temporal expression and localization of
ageing biomarkers of subcellular size and can be conveniently
adapted to image a wide range of small model organisms and
tissue samples.
BibTeX:
@article{Rieckher2015-th,
  author = {Rieckher, Matthias and Kyparissidis-Kokkinidis, Ilias and
Zacharopoulos, Athanasios and Kourmoulakis, Georgios and
Tavernarakis, Nektarios and Ripoll, Jorge and Zacharakis,
Giannis}, title = {A customized light sheet microscope to measure spatio-temporal
protein dynamics in small model organisms}, journal = {PLoS One}, year = {2015}, volume = {10}, number = {5}, pages = {e0127869}, url = {http://dx.doi.org/10.1371/journal.pone.0127869}, doi = {http://dx.doi.org/10.1371/journal.pone.0127869} }
Ritter, Jö.G., Spille, J.-H., Kaminski, T. and Kubitscheck, U. A cylindrical zoom lens unit for adjustable optical sectioning
in light sheet microscopy
2010 Biomed. Opt. Express
Vol. 2(1), pp. 185-193 
article DOI URL 
Abstract: Light sheet microscopy became a powerful tool in life
sciences. Often, however, the sheet geometry is fixed, whereas
it would be advantageous to adjust the sheet geometry to
specimens of different dimensions. Therefore we developed an
afocal cylindrical zoom lens system comprising only 5 lenses
and a total system length of less than 160 mm. Two movable
optical elements were directly coupled, so that the zoom
factor could be adjusted from 1x to 6.3x by a single motor.
Using two different illumination objectives we achieved a
light sheet thickness ranging from 2.4 µm to 36 µm
corresponding to lateral fields of 54 µm to 12.3 mm,
respectively. Polytene chromosomes of salivary gland cell
nuclei of C.tentans larvae were imaged in vivo to demonstrate
the advantages in image contrast by imaging with different
light sheet dimensions.
BibTeX:
@article{Ritter2010-kd,
  author = {Ritter, Jörg G and Spille, Jan-Hendrik and Kaminski, Tim
and Kubitscheck, Ulrich}, title = {A cylindrical zoom lens unit for adjustable optical sectioning
in light sheet microscopy}, journal = {Biomed. Opt. Express}, year = {2010}, volume = {2}, number = {1}, pages = {185--193}, url = {http://dx.doi.org/10.1364/BOE.2.000185}, doi = {http://dx.doi.org/10.1364/BOE.2.000185} }
Ritter, Jö.G., Veith, R., Siebrasse, J.-P. and Kubitscheck, U. High-contrast single-particle tracking by selective focal
plane illumination microscopy
2008 Opt. Express
Vol. 16(10), pp. 7142-7152 
article URL 
Abstract: Wide-field single molecule microscopy is a versatile tool for
analyzing dynamics and molecular interactions in biological
systems. In extended three-dimensional systems, however, the
method suffers from intrinsic out-of-focus fluorescence. We
constructed a high-resolution selective plane illumination
microscope (SPIM) to efficiently solve this problem. The
instrument is an optical sectioning microscope featuring the
high speed and high sensitivity of a video microscope. We
present theoretical calculations and quantitative measurements
of the illumination light sheet thickness yielding 1.7 microm
(FWHM) at 543 nm, 2.0 microm at 633 nm, and a FWHM of the
axial point spread function of 1.13 microm. A direct
comparison of selective plane and epi-illumination of model
samples with intrinsic background fluorescence illustrated the
clear advantage of SPIM for such samples. Single fluorescent
quantum dots in aqueous solution are readily visualized and
tracked proving the suitability of our setup for the study of
fast and dynamic processes in spatially extended biological
specimens.
BibTeX:
@article{Ritter2008-pv,
  author = {Ritter, Jörg G and Veith, Roman and Siebrasse, Jan-Peter
and Kubitscheck, Ulrich}, title = {High-contrast single-particle tracking by selective focal
plane illumination microscopy}, journal = {Opt. Express}, year = {2008}, volume = {16}, number = {10}, pages = {7142--7152}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18545417} }
Ritter, Jö.G., Veith, R., Veenendaal, A., Siebrasse, J.P. and Kubitscheck, U. Light sheet microscopy for single molecule tracking in living
tissue
2010 PLoS One
Vol. 5(7), pp. e11639 
article DOI URL 
Abstract: Single molecule observation in cells and tissue allows the
analysis of physiological processes with molecular detail, but
it still represents a major methodological challenge. Here we
introduce a microscopic technique that combines light sheet
optical sectioning microscopy and ultra sensitive high-speed
imaging. By this approach it is possible to observe single
fluorescent biomolecules in solution, living cells and even
tissue with an unprecedented speed and signal-to-noise ratio
deep within the sample. Thereby we could directly observe and
track small and large tracer molecules in aqueous solution.
Furthermore, we demonstrated the feasibility to visualize the
dynamics of single tracer molecules and native messenger
ribonucleoprotein particles (mRNPs) in salivary gland cell
nuclei of Chironomus tentans larvae up to 200 microm within
the specimen with an excellent signal quality. Thus single
molecule light sheet based fluorescence microscopy allows
analyzing molecular diffusion and interactions in complex
biological systems.
BibTeX:
@article{Ritter2010-lo,
  author = {Ritter, Jörg Gerhard and Veith, Roman and Veenendaal,
Andreas and Siebrasse, Jan Peter and Kubitscheck, Ulrich}, title = {Light sheet microscopy for single molecule tracking in living
tissue}, journal = {PLoS One}, publisher = {Public Library of Science}, year = {2010}, volume = {5}, number = {7}, pages = {e11639}, url = {http://dx.doi.org/10.1371/journal.pone.0011639}, doi = {http://dx.doi.org/10.1371/journal.pone.0011639} }
Rohrbach, A. Artifacts resulting from imaging in scattering media: a
theoretical prediction
2009 Opt. Lett.
Vol. 34(19), pp. 3041-3043 
article DOI URL 
Abstract: Scattering of illumination light from a laser is a severe
problem especially when imaging in thick media. Although this
effect occurs in nearly every imaging process, it can be well
perceived and analyzed in configurations where the optical
axes for illumination and detection are perpendicular to each
other. In this paper I present a theoretical perspective of
how to extend the point-spread function arithmetic from ideal
imaging to realistic imaging including ghost images. These
ghost images are generated by scattered light and are
low-correlated with the ideal image. Numerical simulations of
the propagation of four different types of illumination beams
through a cluster of spheres illustrate the effects of
inhomogeneous object illumination. Clear differences between a
conventional plane-wave illumination, a static light-sheet,
and a laterally scanned Gaussian beam, but also relative to a
scanned Bessel beam, can be observed.
BibTeX:
@article{Rohrbach2009-pl,
  author = {Rohrbach, Alexander},
  title = {Artifacts resulting from imaging in scattering media: a
theoretical prediction}, journal = {Opt. Lett.}, year = {2009}, volume = {34}, number = {19}, pages = {3041--3043}, url = {http://dx.doi.org/10.1364/OL.34.003041}, doi = {http://dx.doi.org/10.1364/OL.34.003041} }
Root, N. Light scanning photomacrography--a brief history and its current
status
1985 J. Biol. Photogr.
Vol. 53(2), pp. 69-77 
article URL 
BibTeX:
@article{Root1985-ax,
  author = {Root, N},
  title = {Light scanning photomacrography--a brief history and its current
status}, journal = {J. Biol. Photogr.}, year = {1985}, volume = {53}, number = {2}, pages = {69--77}, url = {http://www.ncbi.nlm.nih.gov/pubmed/4055656} }
Rosquete, M.R., von Wangenheim, D., Marhavý, P., Barbez, E., Stelzer, E.H.K., Benková, E., Maizel, A. and Kleine-Vehn, Jü. An auxin transport mechanism restricts positive
orthogravitropism in lateral roots.
2013 Curr. Biol.
Vol. 23(9), pp. 817-822 
article  
BibTeX:
@article{Rosquete2013-ad,
  author = {Rosquete, Michel Ruiz and von Wangenheim, Daniel and Marhavý,
Peter and Barbez, Elke and Stelzer, Ernst H K and Benková,
Eva and Maizel, Alexis and Kleine-Vehn, Jürgen}, title = {An auxin transport mechanism restricts positive
orthogravitropism in lateral roots.}, journal = {Curr. Biol.}, year = {2013}, volume = {23}, number = {9}, pages = {817--822} }
Royer, L.A., Weigert, M., Günther, U., Maghelli, N., Jug, F., Sbalzarini, I.F. and Myers, E.W. ClearVolume: open-source live 3D visualization for
light-sheet microscopy
2015 Nat. Methods
Vol. 12(6), pp. 480-481 
article DOI URL 
BibTeX:
@article{Royer2015-mu,
  author = {Royer, Loic A and Weigert, Martin and Günther, Ulrik and
Maghelli, Nicola and Jug, Florian and Sbalzarini, Ivo F and
Myers, Eugene W}, title = {ClearVolume: open-source live 3D visualization for
light-sheet microscopy}, journal = {Nat. Methods}, year = {2015}, volume = {12}, number = {6}, pages = {480--481}, url = {http://dx.doi.org/10.1038/nmeth.3372}, doi = {http://dx.doi.org/10.1038/nmeth.3372} }
Rozbicki, E., Chuai, M., Karjalainen, A.I., Song, F., Sang, H.M., Martin, R., Knölker, H.-J., MacDonald, M.P. and Weijer, C.J. Myosin-II-mediated cell shape changes and cell intercalation
contribute to primitive streak formation
2015 Nat. Cell Biol.
Vol. 17(4), pp. 397-408 
article DOI URL 
Abstract: Primitive streak formation in the chick embryo involves
large-scale highly coordinated flows of more than 100,000
cells in the epiblast. These large-scale tissue flows and
deformations can be correlated with specific anisotropic cell
behaviours in the forming mesendoderm through a combination of
light-sheet microscopy and computational analysis. Relevant
behaviours include apical contraction, elongation along the
apical-basal axis followed by ingression, and asynchronous
directional cell intercalation of small groups of mesendoderm
cells. Cell intercalation is associated with sequential,
directional contraction of apical junctions, the onset,
localization and direction of which correlate strongly with
the appearance of active myosin II cables in aligned apical
junctions in neighbouring cells. Use of class specific myosin
inhibitors and gene-specific knockdown shows that apical
contraction and intercalation are myosin II dependent and also
reveal critical roles for myosin I and myosin V family members
in the assembly of junctional myosin II cables.
BibTeX:
@article{Rozbicki2015-jw,
  author = {Rozbicki, Emil and Chuai, Manli and Karjalainen, Antti I and
Song, Feifei and Sang, Helen M and Martin, René and
Knölker, Hans-Joachim and MacDonald, Michael P and
Weijer, Cornelis J}, title = {Myosin-II-mediated cell shape changes and cell intercalation
contribute to primitive streak formation}, journal = {Nat. Cell Biol.}, year = {2015}, volume = {17}, number = {4}, pages = {397--408}, url = {http://dx.doi.org/10.1038/ncb3138}, doi = {http://dx.doi.org/10.1038/ncb3138} }
Rubio-Guivernau, J.L., Gurchenkov, V., Luengo-Oroz, M.A., Duloquin, L., Bourgine, P., Santos, A., Peyrieras, N. and Ledesma-Carbayo, M.J. Wavelet-based image fusion in multi-view three-dimensional
microscopy
2012 Bioinformatics
Vol. 28(2), pp. 238-245 
article DOI URL 
Abstract: MOTIVATION: Multi-view microscopy techniques such as
Light-Sheet Fluorescence Microscopy (LSFM) are powerful tools
for 3D + time studies of live embryos in developmental
biology. The sample is imaged from several points of view,
acquiring a set of 3D views that are then combined or fused in
order to overcome their individual limitations. Views fusion
is still an open problem despite recent contributions in the
field. RESULTS: We developed a wavelet-based multi-view fusion
method that, due to wavelet decomposition properties, is able
to combine the complementary directional information from all
available views into a single volume. Our method is
demonstrated on LSFM acquisitions from live sea urchin and
zebrafish embryos. The fusion results show improved overall
contrast and details when compared with any of the acquired
volumes. The proposed method does not need knowledge of the
system's point spread function (PSF) and performs better than
other existing PSF independent fusion methods. AVAILABILITY
AND IMPLEMENTATION: The described method was implemented in
Matlab (The Mathworks, Inc., USA) and a graphic user interface
was developed in Java. The software, together with two sample
datasets, is available at
http://www.die.upm.es/im/software/SPIMFusionGUI.zip A public
release, free of charge for non-commercial use, is planned
after the publication of this article.
BibTeX:
@article{Rubio-Guivernau2012-vz,
  author = {Rubio-Guivernau, Jose L and Gurchenkov, Vasily and
Luengo-Oroz, Miguel A and Duloquin, Louise and Bourgine, Paul
and Santos, Andres and Peyrieras, Nadine and Ledesma-Carbayo,
Maria J}, title = {Wavelet-based image fusion in multi-view three-dimensional
microscopy}, journal = {Bioinformatics}, year = {2012}, volume = {28}, number = {2}, pages = {238--245}, url = {http://dx.doi.org/10.1093/bioinformatics/btr609}, doi = {http://dx.doi.org/10.1093/bioinformatics/btr609} }
Saetzler, K. and Eils, R. Resolution improvement by 3D reconstructions from tilted views
in axial tomography and confocal łdots
1997 Bioimaging  article URL 
Abstract: Page 1. 5 (1997) 171--182. Printed in the UK PII:
S0966-9051(97)86221- 1 Resolution improvement by 3-D 171 Page 2.
K Satzler and
BibTeX:
@article{Saetzler1997-bp,
  author = {Saetzler, K and Eils, Roland},
  title = {Resolution improvement by 3D reconstructions from tilted views
in axial tomography and confocal łdots}, journal = {Bioimaging}, year = {1997}, url = {http://doi.wiley.com/10.1002/1361-6374(199712)5:4%3C171::AID-BIO1%3E3.0.CO;2-K} }
Saghafi, S., Becker, K., Hahn, C. and Dodt, H.-U. 3D-ultramicroscopy utilizing aspheric optics 2014 J. Biophotonics
Vol. 7(1-2), pp. 117-125 
article DOI URL 
Abstract: Using a combination of achromatic aspheric optical elements
and achromatic cylindrical lenses, we developed an improved
laser light sheet generator for two-side illumination
ultramicroscopy. This light sheet generator has a much longer
Rayleigh range, a more uniform spatial intensity distribution
along x -, y - and z-axis, and reduced aberrations than the
standard system consisting of a slit aperture and a single
cylindrical lens, which is commonly used in light sheet
microscopy. As there is no truncation of the beam by a slit
aperture in our design the laser energy is used more
efficiently. Applying this light sheet generator to
ultramicroscopy of chemically cleared biological samples, such
as Drosophila, dissected mouse hippocampi, and entire mouse
brains, we achieved a markedly improved resolution of fine
details.
BibTeX:
@article{Saghafi2014-jj,
  author = {Saghafi, Saiedeh and Becker, Klaus and Hahn, Christian and
Dodt, Hans-Ulrich}, title = {3D-ultramicroscopy utilizing aspheric optics}, journal = {J. Biophotonics}, year = {2014}, volume = {7}, number = {1-2}, pages = {117--125}, url = {http://dx.doi.org/10.1002/jbio.201300048}, doi = {http://dx.doi.org/10.1002/jbio.201300048} }
Saghafi, S., Becker, K., Jährling, N., Hahn, C. and Dodt, H.-U. Recent developments in light sheet ultramicroscopy imaging
techniques
2015 European Conferences on Biomedical Optics, pp. 95360C-95360C-7  inproceedings DOI URL 
BibTeX:
@inproceedings{Saghafi2015-ql,
  author = {Saghafi, Saiedeh and Becker, Klaus and Jährling, Nina and
Hahn, Christian and Dodt, Hans-Ulrich}, title = {Recent developments in light sheet ultramicroscopy imaging
techniques}, booktitle = {European Conferences on Biomedical Optics}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {95360C--95360C--7}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2397932}, doi = {http://dx.doi.org/10.1117/12.2184919} }
Saghafi, S., Becker, K., Jährling, N., Richter, M., Kramer, E.R. and Dodt, H.-U. Image enhancement in ultramicroscopy by improved laser light
sheets
2010 J. Biophotonics
Vol. 3(10-11), pp. 686-695 
article DOI URL 
Abstract: In the majority of implementations of light sheet microscopy,
such as ultramicroscopy, the laser beam illuminating the
specimen is truncated by a slit aperture before it is focused
to a light sheet by a single cylindrical lens. A light sheet
generated in this way can be made very thin near to the focal
point, but unfortunately its Rayleigh range is severely
limited. This problem can be partially solved by using a
smaller slit aperture. However, this also causes a major loss
in power, a severe broadening of the beam waist, and thus a
significant loss of resolution along the detection axis. We
developed improved light-sheet-generation optics, which
provide longer Raleigh ranges, whilst retaining beam waists
comparable to our standard system with one cylindrical lens.
Using the modified system we achieved a marked improvement in
the resolution of ultramicroscopy reconstructions of
representative biological specimens.
BibTeX:
@article{Saghafi2010-eu,
  author = {Saghafi, Saiedeh and Becker, Klaus and Jährling, Nina
and Richter, Melanie and Kramer, Edgar R and Dodt, Hans-Ulrich}, title = {Image enhancement in ultramicroscopy by improved laser light
sheets}, journal = {J. Biophotonics}, year = {2010}, volume = {3}, number = {10-11}, pages = {686--695}, url = {http://dx.doi.org/10.1002/jbio.201000047}, doi = {http://dx.doi.org/10.1002/jbio.201000047} }
Samsonov, D., Elsaesser, A., Edwards, A., Thomas, H.M. and Morfill, G.E. High speed laser tomography system 2008 Rev. Sci. Instrum.
Vol. 79(3), pp. 035102 
article DOI URL 
Abstract: A high speed laser tomography system was developed capable of
acquiring three-dimensional (3D) images of optically thin
clouds of moving micron-sized particles. It operates by
parallel-shifting an illuminating laser sheet with a pair of
galvanometer-driven mirrors and synchronously recording
two-dimensional (2D) images of thin slices of the imaged
volume. The maximum scanning speed achieved was 120,000
slices/s, sequences of 24 volume scans (up to 256 slices each)
have been obtained. The 2D slices were stacked to form 3D
images of the volume, then the positions of the particles were
identified and followed in the consecutive scans. The system
was used to image a complex plasma with particles moving at
speeds up to cm/s.
BibTeX:
@article{Samsonov2008-zc,
  author = {Samsonov, D and Elsaesser, A and Edwards, A and Thomas, H M
and Morfill, G E}, title = {High speed laser tomography system}, journal = {Rev. Sci. Instrum.}, year = {2008}, volume = {79}, number = {3}, pages = {035102}, url = {http://dx.doi.org/10.1063/1.2885683}, doi = {http://dx.doi.org/10.1063/1.2885683} }
Santi, P.A. Light sheet fluorescence microscopy: a review 2011 J. Histochem. Cytochem.
Vol. 59(2), pp. 129-138 
article DOI URL 
Abstract: Light sheet fluorescence microscopy (LSFM) functions as a
non-destructive microtome and microscope that uses a plane of
light to optically section and view tissues with subcellular
resolution. This method is well suited for imaging deep within
transparent tissues or within whole organisms, and because
tissues are exposed to only a thin plane of light, specimen
photobleaching and phototoxicity are minimized compared to
wide-field fluorescence, confocal, or multiphoton microscopy.
LSFMs produce well-registered serial sections that are
suitable for three-dimensional reconstruction of tissue
structures. Because of a lack of a commercial LSFM microscope,
numerous versions of light sheet microscopes have been
constructed by different investigators. This review describes
development of the technology, reviews existing devices,
provides details of one LSFM device, and shows examples of
images and three-dimensional reconstructions of tissues that
were produced by LSFM.
BibTeX:
@article{Santi2011-um,
  author = {Santi, Peter A},
  title = {Light sheet fluorescence microscopy: a review},
  journal = {J. Histochem. Cytochem.},
  year = {2011},
  volume = {59},
  number = {2},
  pages = {129--138},
  url = {http://dx.doi.org/10.1369/0022155410394857},
  doi = {http://dx.doi.org/10.1369/0022155410394857}
}
Santi, P.A., Johnson, S.B., Hillenbrand, M., GrandPre, P.Z., Glass, T.J. and Leger, J.R. Thin-sheet laser imaging microscopy for optical sectioning of
thick tissues
2009 Biotechniques
Vol. 46(4), pp. 287-294 
article DOI URL 
Abstract: We report the development of a modular and optimized
thin-sheet laser imaging microscope (TSLIM) for nondestructive
optical sectioning of organisms and thick tissues such as the
mouse cochlea, zebrafish brain/inner ear, and rat brain at a
resolution that is comparable to wide-field fluorescence
microscopy. TSLIM optically sections tissue using a thin sheet
of light by inducing a plane of fluorescence in transparent or
fixed and cleared tissues. Moving the specimen through the
thinnest portion of the light sheet and stitching these image
columns together results in optimal resolution and focus
across the width of a large specimen. Dual light sheets and
aberration-corrected objectives provide uniform section
illumination and reduce absorption artifacts that are common
in light-sheet microscopy. Construction details are provided
for duplication of a TSLIM device by other investigators in
order to encourage further use and development of this
important technology.
BibTeX:
@article{Santi2009-te,
  author = {Santi, Peter A and Johnson, Shane B and Hillenbrand, Matthias
and GrandPre, Patrick Z and Glass, Tiffany J and Leger, James
R}, title = {Thin-sheet laser imaging microscopy for optical sectioning of
thick tissues}, journal = {Biotechniques}, year = {2009}, volume = {46}, number = {4}, pages = {287--294}, url = {http://dx.doi.org/10.2144/000113087}, doi = {http://dx.doi.org/10.2144/000113087} }
Schacht, P., Johnson, S.B. and Santi, P.A. Implementation of a continuous scanning procedure and a line scan
camera for thin-sheet laser imaging microscopy
2010 Biomed. Opt. Express
Vol. 1(2), pp. 598-609 
article DOI URL 
Abstract: We report development of a continuous scanning procedure and the
use of a time delay integration (TDI) line scan camera for a
light-sheet based microscope called a thin-sheet laser imaging
microscope (TSLIM). TSLIM is an optimized version of a
light-sheet fluorescent microscope that previously used a
start/stop scanning procedure to move the specimen through the
thinnest portion of a light-sheet and stitched the image columns
together to produce a well-focused composite image. In this
paper, hardware and software enhancements to TSLIM are described
that allow for dual sided, dual illumination lasers, and
continuous scanning of the specimen using either a full-frame CCD
camera and a TDI line scan camera. These enhancements provided a
~70% reduction in the time required for composite image
generation and a ~63% reduction in photobleaching of the
specimen compared to the start/stop procedure.
BibTeX:
@article{Schacht2010-ol,
  author = {Schacht, Peter and Johnson, Shane B and Santi, Peter A},
  title = {Implementation of a continuous scanning procedure and a line scan
camera for thin-sheet laser imaging microscopy}, journal = {Biomed. Opt. Express}, year = {2010}, volume = {1}, number = {2}, pages = {598--609}, url = {http://dx.doi.org/10.1364/BOE.1.000598/}, doi = {http://dx.doi.org/10.1364/BOE.1.000598} }
Scherf, N. and Huisken, J. The smart and gentle microscope 2015 Nat. Biotechnol.
Vol. 33(8), pp. 815-818 
article DOI URL 
BibTeX:
@article{Scherf2015-qt,
  author = {Scherf, Nico and Huisken, Jan},
  title = {The smart and gentle microscope},
  journal = {Nat. Biotechnol.},
  year = {2015},
  volume = {33},
  number = {8},
  pages = {815--818},
  url = {http://dx.doi.org/10.1038/nbt.3310},
  doi = {http://dx.doi.org/10.1038/nbt.3310}
}
Scherz, P.J., Huisken, J., Sahai-Hernandez, P. and Stainier, D.Y.R. High-speed imaging of developing heart valves reveals
interplay of morphogenesis and function
2008 Development
Vol. 135(6), pp. 1179-1187 
article DOI URL 
Abstract: Knowing how mutations disrupt the interplay between
atrioventricular valve (AVV) morphogenesis and function is
crucial for understanding how congenital valve defects arise.
Here, we use high-speed fluorescence microscopy to investigate
AVV morphogenesis in zebrafish at cellular resolution. We find
that valve leaflets form directly through a process of
invagination, rather than first forming endocardial cushions.
There are three phases of valve function in embryonic
development. First, the atrioventricular canal (AVC) is closed
by the mechanical action of the myocardium, rolls together and
then relaxes. The growing valve leaflets serve to block the
canal during the roll and, depending on the developmental
stage, either expand or hang down as a leaflet to block the
canal. These steps are disrupted by the subtle morphological
changes that result from inhibiting ErbB-, TGFbeta-or Cox2
(Ptgs2)-dependent signaling. Cox2 inhibition affects valve
development due to its effect on myocardial cell size and
shape, which changes the morphology of the ventricle and
alters valve geometry. Thus, different signaling pathways
regulate distinct aspects of the behavior of individual cells
during valve morphogenesis, thereby influencing specific
facets of valve function.
BibTeX:
@article{Scherz2008-wb,
  author = {Scherz, Paul J and Huisken, Jan and Sahai-Hernandez, Pankaj
and Stainier, Didier Y R}, title = {High-speed imaging of developing heart valves reveals
interplay of morphogenesis and function}, journal = {Development}, year = {2008}, volume = {135}, number = {6}, pages = {1179--1187}, url = {http://dx.doi.org/10.1242/dev.010694}, doi = {http://dx.doi.org/10.1242/dev.010694} }
Scheul, T., Wang, I. and Vial, J.-C. STED-SPIM made simple 2014 Opt. Express
Vol. 22(25), pp. 30852-30864 
article DOI URL 
Abstract: We report the development of a stimulated emission depletion
(STED) selective plane illumination (SPIM) microscope based on a
single diode-pumped solid state (DPSS) laser that simultaneously
delivers nanosecond-pulses at two wavelengths. The two
wavelengths, 355 nm and 532 nm, are generated by harmonic
conversion and they are used to induce respectively excitation
and stimulated emission depletion. This source should allow a
low-cost, compact, very efficient and simplified STED scheme
since the two beams are intrinsically aligned and synchronized.
Using a chromatic beam shaping device which leaves the
excitation beam unaffected and produces a donut-shaped STED
beam, we demonstrate a 300% reduction of the light sheet
thickness, together with an enhancement of the sheet uniformity
over larger field of view, at low STED power, in Coumarin dye
solution.
BibTeX:
@article{Scheul2014-zf,
  author = {Scheul, Teodora and Wang, Irène and Vial, Jean-Claude},
  title = {STED-SPIM made simple},
  journal = {Opt. Express},
  publisher = {Optical Society of America},
  year = {2014},
  volume = {22},
  number = {25},
  pages = {30852--30864},
  url = {http://dx.doi.org/10.1364/OE.22.030852},
  doi = {http://dx.doi.org/10.1364/OE.22.030852}
}
Schickinger, S., Bruns, T., Wittig, R., Weber, P., Wagner, M. and Schneckenburger, H. Nanosecond ratio imaging of redox states in tumor cell
spheroids using light sheet-based fluorescence microscopy
2013 J. Biomed. Opt.
Vol. 18(12), pp. 126007 
article DOI URL 
Abstract: A new concept of three-dimensional imaging of tumor cell
spheroids by light sheet-based fluorescence microscopy and
nanosecond ratio imaging is described. Due to its low light
dose and alternative excitation by two laser wavelengths (391
and 470 nm), this method maintains cell viability and permits
recording of real-time kinetics. A genetically encoded sensor
permits measurement of the redox state of glutathione and
visualization of the impact of oxygen radicals. The
pharmaceutically relevant system is tested upon addition of an
oxidizing agent (H2O2), as well as upon addition of the
apoptosis-inducing agent staurosporine.
BibTeX:
@article{Schickinger2013-fi,
  author = {Schickinger, Sarah and Bruns, Thomas and Wittig, Rainer and
Weber, Petra and Wagner, Michael and Schneckenburger, Herbert}, title = {Nanosecond ratio imaging of redox states in tumor cell
spheroids using light sheet-based fluorescence microscopy}, journal = {J. Biomed. Opt.}, year = {2013}, volume = {18}, number = {12}, pages = {126007}, url = {http://dx.doi.org/10.1117/1.JBO.18.12.126007}, doi = {http://dx.doi.org/10.1117/1.JBO.18.12.126007} }
Schmid, B. and Huisken, J. Real-time multi-view deconvolution 2015 Bioinformatics  article DOI URL 
Abstract: In light-sheet microscopy, overall image content and
resolution are improved by acquiring and fusing multiple views
of the sample from different directions. State-of-the-art
multi-view (MV) deconvolution simultaneously fuses and
deconvolves the images in 3D, but processing takes a multiple
of the acquisition time and constitutes the bottleneck in the
imaging pipeline. Here, we show that MV deconvolution in 3D
can finally be achieved in real-time by processing
cross-sectional planes individually on the massively parallel
architecture of a graphics processing unit (GPU). Our
approximation is valid in the typical case where the rotation
axis lies in the imaging plane. AVAILABILITY AND
IMPLEMENTATION: Source code and binaries are available on
github (https://github.com/bene51/), native code under the
repository 'gpudeconvolution', Java wrappers implementing
Fiji plugins under 'SPIMReconstructionCuda'. CONTACT:
bschmid@mpi-cbg.de or huisken@mpi-cbg.deSupplementary
information: Supplementary data are available at
Bioinformatics online.
BibTeX:
@article{Schmid2015-ht,
  author = {Schmid, Benjamin and Huisken, Jan},
  title = {Real-time multi-view deconvolution},
  journal = {Bioinformatics},
  year = {2015},
  url = {http://dx.doi.org/10.1093/bioinformatics/btv387},
  doi = {http://dx.doi.org/10.1093/bioinformatics/btv387}
}
Schmid, B., Shah, G., Scherf, N., Weber, M., Thierbach, K., Campos, C.P., Roeder, I., Aanstad, P. and Huisken, J. High-speed panoramic light-sheet microscopy reveals global
endodermal cell dynamics
2013 Nat. Commun.
Vol. 4, pp. 2207 
article DOI URL 
Abstract: The ever-increasing speed and resolution of modern microscopes
make the storage and post-processing of images challenging and
prevent thorough statistical analyses in developmental
biology. Here, instead of deploying massive storage and
computing power, we exploit the spherical geometry of
zebrafish embryos by computing a radial maximum intensity
projection in real time with a 240-fold reduction in data
rate. In our four-lens selective plane illumination microscope
(SPIM) setup the development of multiple embryos is recorded
in parallel and a map of all labelled cells is obtained for
each embryo in <10 s. In these panoramic projections, cell
segmentation and flow analysis reveal characteristic migration
patterns and global tissue remodelling in the early endoderm.
Merging data from many samples uncover stereotypic patterns
that are fundamental to endoderm development in every embryo.
We demonstrate that processing and compressing raw image data
in real time is not only efficient but indispensable for
image-based systems biology.
BibTeX:
@article{Schmid2013-xu,
  author = {Schmid, Benjamin and Shah, Gopi and Scherf, Nico and Weber,
Michael and Thierbach, Konstantin and Campos, Citlali
Pérez and Roeder, Ingo and Aanstad, Pia and Huisken, Jan}, title = {High-speed panoramic light-sheet microscopy reveals global
endodermal cell dynamics}, journal = {Nat. Commun.}, year = {2013}, volume = {4}, pages = {2207}, url = {http://dx.doi.org/10.1038/ncomms3207}, doi = {http://dx.doi.org/10.1038/ncomms3207} }
Schmied, C., Stamataki, E. and Tomancak, P. Open-source solutions for SPIMage processing 2014 Methods Cell Biol.
Vol. 123, pp. 505-529 
article DOI URL 
Abstract: Light sheet microscopy is an emerging technique allowing
comprehensive visualization of dynamic biological processes,
at high spatial and temporal resolution without significant
damage to the sample by the imaging process itself. It thus
lends itself to time-lapse observation of fluorescently
labeled molecular markers over long periods of time in a
living specimen. In combination with sample rotation light
sheet microscopy and in particular its selective plane
illumination microscopy (SPIM) flavor, enables imaging of
relatively large specimens, such as embryos of animal model
organisms, in their entirety. The benefits of SPIM multiview
imaging come to the cost of image data postprocessing
necessary to deliver the final output that can be analyzed.
Here, we provide a set of practical recipes that walk
biologists through the complex processes of SPIM data
registration, fusion, deconvolution, and time-lapse
registration using publicly available open-source tools. We
explain, in plain language, the basic principles behind SPIM
image-processing algorithms that should enable users to make
informed decisions during parameter tuning of the various
processing steps applied to their own datasets. Importantly,
the protocols presented here are applicable equally to
processing of multiview SPIM data from the commercial Zeiss
Lightsheet Z.1 microscope and from the open-access SPIM
platforms such as OpenSPIM.
BibTeX:
@article{Schmied2014-tz,
  author = {Schmied, Christopher and Stamataki, Evangelia and Tomancak,
Pavel}, title = {Open-source solutions for SPIMage processing}, journal = {Methods Cell Biol.}, year = {2014}, volume = {123}, pages = {505--529}, url = {http://dx.doi.org/10.1016/B978-0-12-420138-5.00027-6}, doi = {http://dx.doi.org/10.1016/B978-0-12-420138-5.00027-6} }
Schneckenburger, H., Weber, P., Schickinger, S., Richter, V., Bruns, T. and Wagner, M. 3D imaging of apoptosis by FRET, light sheet fluorescence
and scattering microsopy
2015 European Conferences on Biomedical Optics, pp. 953716-953716-5  inproceedings DOI URL 
BibTeX:
@inproceedings{Schneckenburger2015-fj,
  author = {Schneckenburger, Herbert and Weber, Petra and Schickinger, Sarah
and Richter, Verena and Bruns, Thomas and Wagner, Michael}, title = {3D imaging of apoptosis by FRET, light sheet fluorescence
and scattering microsopy}, booktitle = {European Conferences on Biomedical Optics}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {953716--953716--5}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2398279}, doi = {http://dx.doi.org/10.1117/12.2183607} }
Schröter, T.J., Johnson, S.B., John, K. and Santi, P.A. Scanning thin-sheet laser imaging microscopy (sTSLIM) with
structured illumination and HiLo background rejection
2012 Biomed. Opt. Express
Vol. 3(1), pp. 170-177 
article DOI URL 
Abstract: We report replacement of one side of a static illumination,
dual sided, thin-sheet laser imaging microscope (TSLIM) with
an intensity modulated laser scanner in order to implement
structured illumination (SI) and HiLo image demodulation
techniques for background rejection. The new system is
equipped with one static and one scanned light-sheet and is
called a scanning thin-sheet laser imaging microscope
(sTSLIM). It is an optimized version of a light-sheet
fluorescent microscope that is designed to image large
specimens (<15 mm in diameter). In this paper we describe the
hardware and software modifications to TSLIM that allow for
static and uniform light-sheet illumination with SI and HiLo
image demodulation. The static light-sheet has a thickness of
3.2 µm; whereas, the scanned side has a light-sheet thickness
of 4.2 µm. The scanned side images specimens with subcellular
resolution (<1 µm lateral and <4 µm axial resolution) with a
size up to 15 mm. SI and HiLo produce superior contrast
compared to both the uniform static and scanned light-sheets.
HiLo contrast was greater than SI and is faster and more
robust than SI because as it produces images in two-thirds of
the time and exhibits fewer intensity streaking artifacts.
BibTeX:
@article{Schroter2012-fw,
  author = {Schröter, Tobias J and Johnson, Shane B and John,
Kerstin and Santi, Peter A}, title = {Scanning thin-sheet laser imaging microscopy (sTSLIM) with
structured illumination and HiLo background rejection}, journal = {Biomed. Opt. Express}, year = {2012}, volume = {3}, number = {1}, pages = {170--177}, url = {http://dx.doi.org/10.1364/BOE.3.000170}, doi = {http://dx.doi.org/10.1364/BOE.3.000170} }
Selchow, O. Von der Seite beleuchtet: Selective PLane Illumination
Microscopy
2006 BIOspektrum
Vol. 12, pp. 3 
article  
BibTeX:
@article{Selchow2006-iv,
  author = {Selchow, Olaf},
  title = {Von der Seite beleuchtet: Selective PLane Illumination
Microscopy}, journal = {BIOspektrum}, year = {2006}, volume = {12}, pages = {3} }
Selchow, O. and Huisken, J. Lichtblattmikroskopie: Das Beleuchtungskonzept revolutioniert die
3D-Analyse lebender Proben
2013 Biophotonik
Vol. 1, pp. 24-27 
article  
BibTeX:
@article{Selchow2013-ih,
  author = {Selchow, Olaf and Huisken, Jan},
  title = {Lichtblattmikroskopie: Das Beleuchtungskonzept revolutioniert die
3D-Analyse lebender Proben}, journal = {Biophotonik}, year = {2013}, volume = {1}, pages = {24--27} }
Sena, G., Frentz, Z., Birnbaum, K.D. and Leibler, S. Quantitation of cellular dynamics in growing Arabidopsis roots
with light sheet microscopy
2011 PLoS One
Vol. 6(6), pp. e21303 
article DOI URL 
Abstract: To understand dynamic developmental processes, living tissues
have to be imaged frequently and for extended periods of time.
Root development is extensively studied at cellular resolution
to understand basic mechanisms underlying pattern formation
and maintenance in plants. Unfortunately, ensuring continuous
specimen access, while preserving physiological conditions and
preventing photo-damage, poses major barriers to measurements
of cellular dynamics in growing organs such as plant roots. We
present a system that integrates optical sectioning through
light sheet fluorescence microscopy with hydroponic culture
that enables us to image, at cellular resolution, a vertically
growing Arabidopsis root every few minutes and for several
consecutive days. We describe novel automated routines to
track the root tip as it grows, to track cellular nuclei and
to identify cell divisions. We demonstrate the system's
capabilities by collecting data on divisions and nuclear
dynamics.
BibTeX:
@article{Sena2011-fe,
  author = {Sena, Giovanni and Frentz, Zak and Birnbaum, Kenneth D and
Leibler, Stanislas}, title = {Quantitation of cellular dynamics in growing Arabidopsis roots
with light sheet microscopy}, journal = {PLoS One}, year = {2011}, volume = {6}, number = {6}, pages = {e21303}, url = {http://dx.doi.org/10.1371/journal.pone.0021303}, doi = {http://dx.doi.org/10.1371/journal.pone.0021303} }
Shaw, P.J., Agard, D.A., Hiraoka, Y. and Sedat, J.W. Tilted view reconstruction in optical microscopy.
Three-dimensional reconstruction of Drosophila melanogaster
embryo nuclei
1989 Biophys. J.
Vol. 55(1), pp. 101-110 
article DOI URL 
Abstract: The resolution along the optical axis (z) is much less than
the in-plane resolution in any current optical microscope,
conventional or otherwise. We have used mutually tilted,
through-focal section views of the same object to provide a
solution to this problem. A tilting specimen stage was
constructed for an optical microscope, which with the use of a
coverslip-free water immersion lens, allowed the collection of
data sets from intact Drosophila melanogaster embryos at
viewing directions up to 90 degrees apart. We have devised an
image processing scheme to determine the relative tilt,
translation, and sampling parameters of the different data
sets. This involves the use of a modified phase
cross-correlation function, which produces a very sharp
maximum. Finally the data sets are merged using
figure-of-merit and local area scaling techniques borrowed
from x-ray protein crystallography. We demonstrate the
application of this technique to data sets from a metaphase
plate in an embryo of Drosophila melanogaster. As expected,
the merged reconstruction combined the highest resolution
available in the individual data sets. As estimated from the
Fourier transform, the final resolution is 0.25 microns in x
and y and 0.4 microns in z. In the final reconstruction all
ten chromosome arms can be easily delineated; this was not
possible in the individual data sets. Within many of the arms
the two individual chromatids can be seen. In some cases the
chromatids are wrapped around each other helically, in others
they lie alongside each other in a parallel arrangement.
BibTeX:
@article{Shaw1989-gh,
  author = {Shaw, P J and Agard, D A and Hiraoka, Y and Sedat, J W},
  title = {Tilted view reconstruction in optical microscopy.
Three-dimensional reconstruction of Drosophila melanogaster
embryo nuclei}, journal = {Biophys. J.}, year = {1989}, volume = {55}, number = {1}, pages = {101--110}, url = {http://dx.doi.org/10.1016/S0006-3495(89)82783-3}, doi = {http://dx.doi.org/10.1016/S0006-3495(89)82783-3} }
Siebrasse, J.P., Kaminski, T. and Kubitscheck, U. Nuclear export of single native mRNA molecules observed by
light sheet fluorescence microscopy
2012 Proc. Natl. Acad. Sci. U. S. A.
Vol. 109(24), pp. 9426-9431 
article DOI URL 
Abstract: Nuclear export of mRNA is a key transport process in
eukaryotic cells. To investigate it, we labeled native mRNP
particles in living Chironomus tentans salivary gland cells
with fluorescent hrp36, the hnRNP A1 homolog, and the nuclear
envelope by fluorescent NTF2. Using light sheet microscopy, we
traced single native mRNA particles across the nuclear
envelope. The particles were observed to often probe nuclear
pore complexes (NPC) at their nuclear face, and in only 25br> of the cases yielded actual export. The complete export
process took between 65 ms up to several seconds. A
rate-limiting step was observed, which could be assigned to
the nuclear basket of the pore and might correspond to a
repositioning and unfolding of mRNPs before the actual
translocation. Analysis of single fluorescent Dbp5 molecules,
the RNA helicase essential for mRNA export, revealed that Dbp5
most often approached the cytoplasmic face of the NPC, and
exhibited a binding duration of approximately 55 ms. Our
results have allowed a refinement of the current models for
mRNA export.
BibTeX:
@article{Siebrasse2012-bn,
  author = {Siebrasse, Jan Peter and Kaminski, Tim and Kubitscheck, Ulrich},
  title = {Nuclear export of single native mRNA molecules observed by
light sheet fluorescence microscopy}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, year = {2012}, volume = {109}, number = {24}, pages = {9426--9431}, url = {http://dx.doi.org/10.1073/pnas.1201781109}, doi = {http://dx.doi.org/10.1073/pnas.1201781109} }
Siedentopf, H. and Zsigmondy, R. Uber Sichtbarmachung und Größenbestimmung
ultramikoskopischer Teilchen, mit besonderer Anwendung auf
Goldrubingläser
1902 Ann. Phys.
Vol. 315(1), pp. 1-39 
article DOI URL 
BibTeX:
@article{Siedentopf1902-xh,
  author = {Siedentopf, H and Zsigmondy, R},
  title = {Uber Sichtbarmachung und Größenbestimmung
ultramikoskopischer Teilchen, mit besonderer Anwendung auf
Goldrubingläser}, journal = {Ann. Phys.}, publisher = {WILEY-VCH Verlag}, year = {1902}, volume = {315}, number = {1}, pages = {1--39}, url = {http://dx.doi.org/10.1002/andp.19023150102}, doi = {http://dx.doi.org/10.1002/andp.19023150102} }
Silvestri, L., Allegra Mascaro, A.L., Costantini, I., Sacconi, L. and Pavone, F.S. Correlative two-photon and light sheet microscopy 2014 Methods
Vol. 66(2), pp. 268-272 
article DOI URL 
Abstract: Information processing inside the central nervous system takes
place on multiple scales in both space and time. A single
imaging technique can reveal only a small part of this complex
machinery. To obtain a more comprehensive view of brain
functionality, complementary approaches should be combined
into a correlative framework. Here, we describe a method to
integrate data from in vivo two-photon fluorescence imaging
and ex vivo light sheet microscopy, taking advantage of blood
vessels as reference chart. We show how the apical dendritic
arbor of a single cortical pyramidal neuron imaged in living
thy1-GFP-M mice can be found in the large-scale brain
reconstruction obtained with light sheet microscopy. Starting
from the apical portion, the whole pyramidal neuron can then
be segmented. The correlative approach presented here allows
contextualizing within a three-dimensional anatomic framework
the neurons whose dynamics have been observed with high detail
in vivo.
BibTeX:
@article{Silvestri2014-nr,
  author = {Silvestri, Ludovico and Allegra Mascaro, Anna Letizia and
Costantini, Irene and Sacconi, Leonardo and Pavone, Francesco
Saverio}, title = {Correlative two-photon and light sheet microscopy}, journal = {Methods}, year = {2014}, volume = {66}, number = {2}, pages = {268--272}, url = {http://dx.doi.org/10.1016/j.ymeth.2013.06.013}, doi = {http://dx.doi.org/10.1016/j.ymeth.2013.06.013} }
Silvestri, L., Bria, A., Costantini, I., Sacconi, L., Peng, H., Iannello, G. and Pavone, F.S. Micron-scale resolution optical tomography of entire mouse
brains with confocal light sheet microscopy
2013 J. Vis. Exp.(80)  article DOI URL 
Abstract: Understanding the architecture of mammalian brain at
single-cell resolution is one of the key issues of
neuroscience. However, mapping neuronal soma and projections
throughout the whole brain is still challenging for imaging
and data management technologies. Indeed, macroscopic volumes
need to be reconstructed with high resolution and contrast in
a reasonable time, producing datasets in the TeraByte range.
We recently demonstrated an optical method (confocal light
sheet microscopy, CLSM) capable of obtaining micron-scale
reconstruction of entire mouse brains labeled with enhanced
green fluorescent protein (EGFP). Combining light sheet
illumination and confocal detection, CLSM allows deep imaging
inside macroscopic cleared specimens with high contrast and
speed. Here we describe the complete experimental pipeline to
obtain comprehensive and human-readable images of entire mouse
brains labeled with fluorescent proteins. The clearing and the
mounting procedures are described, together with the steps to
perform an optical tomography on its whole volume by acquiring
many parallel adjacent stacks. We showed the usage of
open-source custom-made software tools enabling stitching of
the multiple stacks and multi-resolution data navigation.
Finally, we illustrated some example of brain maps: the
cerebellum from an L7-GFP transgenic mouse, in which all
Purkinje cells are selectively labeled, and the whole brain
from a thy1-GFP-M mouse, characterized by a random sparse
neuronal labeling.
BibTeX:
@article{Silvestri2013-zc,
  author = {Silvestri, Ludovico and Bria, Alessandro and Costantini, Irene
and Sacconi, Leonardo and Peng, Hanchuan and Iannello, Giulio
and Pavone, Francesco Saverio}, title = {Micron-scale resolution optical tomography of entire mouse
brains with confocal light sheet microscopy}, journal = {J. Vis. Exp.}, year = {2013}, number = {80}, url = {http://dx.doi.org/10.3791/50696}, doi = {http://dx.doi.org/10.3791/50696} }
Silvestri, L., Bria, A., Sacconi, L., Iannello, G. and Pavone, F.S. Confocal light sheet microscopy: micron-scale neuroanatomy of
the entire mouse brain
2012 Opt. Express
Vol. 20(18), pp. 20582-20598 
article DOI URL 
Abstract: Elucidating the neural pathways that underlie brain function
is one of the greatest challenges in neuroscience. Light sheet
based microscopy is a cutting edge method to map cerebral
circuitry through optical sectioning of cleared mouse brains.
However, the image contrast provided by this method is not
sufficient to resolve and reconstruct the entire neuronal
network. Here we combined the advantages of light sheet
illumination and confocal slit detection to increase the image
contrast in real time, with a frame rate of 10 Hz. In fact, in
confocal light sheet microscopy (CLSM), the out-of-focus and
scattered light is filtered out before detection, without
multiple acquisitions or any post-processing of the acquired
data. The background rejection capabilities of CLSM were
validated in cleared mouse brains by comparison with a
structured illumination approach. We show that CLSM allows
reconstructing macroscopic brain volumes with sub-cellular
resolution. We obtained a comprehensive map of Purkinje cells
in the cerebellum of L7-GFP transgenic mice. Further, we were
able to trace neuronal projections across brain of thy1-GFP-M
transgenic mice. The whole-brain high-resolution fluorescence
imaging assured by CLSM may represent a powerful tool to
navigate the brain through neuronal pathways. Although this
work is focused on brain imaging, the macro-scale
high-resolution tomographies affordable with CLSM are ideally
suited to explore, at micron-scale resolution, the anatomy of
different specimens like murine organs, embryos or flies.
BibTeX:
@article{Silvestri2012-ys,
  author = {Silvestri, L and Bria, A and Sacconi, L and Iannello, G and
Pavone, F S}, title = {Confocal light sheet microscopy: micron-scale neuroanatomy of
the entire mouse brain}, journal = {Opt. Express}, year = {2012}, volume = {20}, number = {18}, pages = {20582--20598}, url = {http://dx.doi.org/10.1364/OE.20.020582}, doi = {http://dx.doi.org/10.1364/OE.20.020582} }
Silvestri, L., Mascaro, A.L.A., Costantini, I., Sacconi, L. and Pavone, F.S. Exploring the brain on multiple scales with correlative
two-photon and light sheet microscopy
2014 SPIE BiOS, pp. 89480I-89480I-6  inproceedings DOI URL 
BibTeX:
@inproceedings{Silvestri2014-lc,
  author = {Silvestri, Ludovico and Mascaro, Anna Letizia Allegra and
Costantini, Irene and Sacconi, Leonardo and Pavone, Francesco S}, title = {Exploring the brain on multiple scales with correlative
two-photon and light sheet microscopy}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {89480I--89480I--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1839935}, doi = {http://dx.doi.org/10.1117/12.2037867} }
Silvestri, L., Paciscopi, M., Soda, P., Biamonte, F., Iannello, G., Frasconi, P. and Pavone, F.S. Quantitative neuroanatomy of all Purkinje cells with light
sheet microscopy and high-throughput image analysis
2015 Front. Neuroanat.
Vol. 9, pp. 68 
article DOI URL 
Abstract: Characterizing the cytoarchitecture of mammalian central
nervous system on a brain-wide scale is becoming a compelling
need in neuroscience. For example, realistic modeling of brain
activity requires the definition of quantitative features of
large neuronal populations in the whole brain. Quantitative
anatomical maps will also be crucial to classify the
cytoarchtitectonic abnormalities associated with neuronal
pathologies in a high reproducible and reliable manner. In
this paper, we apply recent advances in optical microscopy and
image analysis to characterize the spatial distribution of
Purkinje cells (PCs) across the whole cerebellum. Light sheet
microscopy was used to image with micron-scale resolution a
fixed and cleared cerebellum of an L7-GFP transgenic mouse, in
which all PCs are fluorescently labeled. A fast and scalable
algorithm for fully automated cell identification was applied
on the image to extract the position of all the fluorescent
PCs. This vectorized representation of the cell population
allows a thorough characterization of the complex
three-dimensional distribution of the neurons, highlighting
the presence of gaps inside the lamellar organization of PCs,
whose density is believed to play a significant role in autism
spectrum disorders. Furthermore, clustering analysis of the
localized somata permits dividing the whole cerebellum in
groups of PCs with high spatial correlation, suggesting new
possibilities of anatomical partition. The quantitative
approach presented here can be extended to study the
distribution of different types of cell in many brain regions
and across the whole encephalon, providing a robust base for
building realistic computational models of the brain, and for
unbiased morphological tissue screening in presence of
pathologies and/or drug treatments.
BibTeX:
@article{Silvestri2015-cy,
  author = {Silvestri, Ludovico and Paciscopi, Marco and Soda, Paolo and
Biamonte, Filippo and Iannello, Giulio and Frasconi, Paolo and
Pavone, Francesco S}, title = {Quantitative neuroanatomy of all Purkinje cells with light
sheet microscopy and high-throughput image analysis}, journal = {Front. Neuroanat.}, year = {2015}, volume = {9}, pages = {68}, url = {http://dx.doi.org/10.3389/fnana.2015.00068}, doi = {http://dx.doi.org/10.3389/fnana.2015.00068} }
Silvestri, L., Sacconi, L. and Pavone, F.S. Correcting spherical aberrations in confocal light sheet
microscopy: a theoretical study
2014 Microsc. Res. Tech.
Vol. 77(7), pp. 483-491 
article DOI URL 
Abstract: In the last years, fluorescence light sheet microscopy has
attracted an increasing interest among the microscopy
community. One of the most promising applications of this
technique is the reconstruction of macroscopic biological
specimens with microscopic resolution, without physical
sectioning. To this aim, light sheet microscopy is combined
with clearing protocols based on refractive index matching,
which render the tissue transparent. However, these protocols
lead to a huge drop in the fluorescence signal, limiting their
practical applicability. The reduction of signal to background
ratio is commonly ascribed to chemical degradation of the
fluorophores by the organic solvents used for clearing. This
view however completely neglects another important factor of
contrast loss, i.e., optical aberrations. In fact,
commercially available objectives suitable for light sheet
microscopy are not designed for the refractive index of the
clearing solutions, and this mismatch introduces severe
spherical aberration. Here we simulated the aberrated point
spread function (PSF) of a light sheet microscope with
confocal slit detection. We investigated the variation of the
PSF as a function of objective numerical aperture (NA) and of
imaging depth inside the clearing solution. We also explored
the possibility of correcting such spherical aberration by
introducing extra optical devices in the detection path. By
correcting up to the second order spherical aberration, a
quasi-diffraction-limited regime can be recovered, and image
quality is restored.
BibTeX:
@article{Silvestri2014-ot,
  author = {Silvestri, L and Sacconi, L and Pavone, F S},
  title = {Correcting spherical aberrations in confocal light sheet
microscopy: a theoretical study}, journal = {Microsc. Res. Tech.}, year = {2014}, volume = {77}, number = {7}, pages = {483--491}, url = {http://dx.doi.org/10.1002/jemt.22330}, doi = {http://dx.doi.org/10.1002/jemt.22330} }
Silvestri, L., Sacconi, L. and Pavone, F.S. Light sheet microscopy of cleared mouse brains: aberrations
effects caused by refractive index mismatch
2013 European Conference on Biomedical Optics, pp. 880405  inproceedings URL 
Abstract: Fluorescence light sheet microscopy has known a true renaissance
in the last years. In fact, since optical sectioning is achieved
in a wide-field detection scheme, this technique allows high
resolution three-dimensional imaging with high frame rate. Light
sheet microscopy is therefore an ideal candidate for
reconstructing macroscopic specimens with micron resolution:
coupled with clearing protocols based on refractive index
matching it has been exploited to image entire mouse brains
without physical sectioning. Use of clearing protocols poses
several challenges to light sheet microscopy. First of all,
residual light scattering inside the tissue expands the
excitation light sheet, leading to the excitation of out-offocus
planes, and thus frustrating the very principle of light sheet
illumination. To reject out-of-focus contributions we recently
coupled light sheet illumination with confocal detection,
achieving significant contrast enhancement in real time. Another
issue which often arises when working with clearing agents is
the refractive index mismatch between the clearing and the
medium objective design medium. This introduces severe spherical
aberration, which leads to broadening of the point spread
function and to a strong reduction in its peak value: When
imaging deep (several mm) inside macroscopic specimens, the
signal can be reduced by more than an order of magnitude. We
investigated the possibility of correcting such spherical
aberration by introducing extra optical devices in the detection
path.
BibTeX:
@inproceedings{Silvestri2013-zt,
  author = {Silvestri, Ludovico and Sacconi, Leonardo and Pavone, Francesco
S}, title = {Light sheet microscopy of cleared mouse brains: aberrations
effects caused by refractive index mismatch}, booktitle = {European Conference on Biomedical Optics}, publisher = {Optical Society of America}, year = {2013}, pages = {880405}, url = {http://www.opticsinfobase.org/abstract.cfm?uri=ECBO-2013-880405} }
Simon, W. Photomicrography of deep fields 1965 Rev. Sci. Instrum.
Vol. 36(11), pp. 1654-1655 
article URL 
BibTeX:
@article{Simon1965-xy,
  author = {Simon, W},
  title = {Photomicrography of deep fields},
  journal = {Rev. Sci. Instrum.},
  year = {1965},
  volume = {36},
  number = {11},
  pages = {1654--1655},
  url = {http://www.ncbi.nlm.nih.gov/pubmed/5832419}
}
Singh, A.P., Krieger, J.W., Buchholz, J., Charbon, E., Langowski, Jö. and Wohland, T. The performance of 2D array detectors for light sheet based
fluorescence correlation spectroscopy
2013 Opt. Express
Vol. 21(7), pp. 8652-8668 
article DOI URL 
Abstract: Single plane illumination microscopy based fluorescence
correlation spectroscopy (SPIM-FCS) is a new method for
imaging FCS in 3D samples, providing diffusion coefficients,
transport, flow velocities and concentrations in an imaging
mode. SPIM-FCS records correlation functions over a whole
plane in a sample, which requires array detectors for
recording the fluorescence signal. Several types of image
sensors are suitable for FCS. They differ in properties such
as effective area per pixel, quantum efficiency, noise level
and read-out speed. Here we compare the performance of several
low light array detectors based on three different
technologies: (1) Single-photon avalanche diode (SPAD) arrays,
(2) passive-pixel electron multiplying charge coupled device
(EMCCD) and (3) active-pixel scientific-grade complementary
metal oxide semiconductor cameras (sCMOS). We discuss the
influence of the detector characteristics on the effective FCS
observation volume, and demonstrate that light sheet based
SPIM-FCS provides absolute diffusion coefficients. This is
verified by parallel measurements with confocal FCS, single
particle tracking (SPT), and the determination of
concentration gradients in space and time. While EMCCD cameras
have a temporal resolution in the millisecond range, sCMOS
cameras and SPAD arrays can extend the time resolution of
SPIM-FCS down to 10 $s or lower.
BibTeX:
@article{Singh2013-aq,
  author = {Singh, Anand Pratap and Krieger, Jan Wolfgang and Buchholz,
Jan and Charbon, Edoardo and Langowski, Jörg and
Wohland, Thorsten}, title = {The performance of 2D array detectors for light sheet based
fluorescence correlation spectroscopy}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {7}, pages = {8652--8668}, url = {http://dx.doi.org/10.1364/OE.21.008652}, doi = {http://dx.doi.org/10.1364/OE.21.008652} }
Sinkó, J., Szabó, G. and Erdélyi, M. Ray tracing analysis of inclined illumination techniques 2014 Opt. Express
Vol. 22(16), pp. 18940-18948 
article DOI URL 
Abstract: The reduction of out of focus signal is a general task in
fluorescence microscopy and is especially important in the
recently developed super-resolution techniques because of the
degradation of the final image. Several illumination methods
have been developed to provide decreased out of focus signal
level relative to the common epifluorescent illumination. In
this paper we examine the highly inclined and the total internal
reflection illumination techniques using the ray tracing method.
Two merit functions were introduced for the quantitative
description of the excitation of the selected region. We studied
the feasibility of illumination methods, and the required
corrections arising from the imperfections of the optical
elements.
BibTeX:
@article{Sinko2014-ls,
  author = {Sinkó, József and Szabó, Gábor and Erdélyi,
Miklós}, title = {Ray tracing analysis of inclined illumination techniques}, journal = {Opt. Express}, publisher = {opticsinfobase.org}, year = {2014}, volume = {22}, number = {16}, pages = {18940--18948}, url = {http://dx.doi.org/10.1364/OE.22.018940}, doi = {http://dx.doi.org/10.1364/OE.22.018940} }
Skupsch, C. and Brücker, C. Multiple-plane particle image velocimetry using a light-field
camera
2013 Opt. Express
Vol. 21(2), pp. 1726-1740 
article DOI URL 
Abstract: Planar velocity fields in flows are determined simultaneously
on parallel measurement planes by means of an in-house
manufactured light-field camera. The planes are defined by
illuminating light sheets with constant spacing. Particle
positions are reconstructed from a single 2D recording taken
by a CMOS-camera equipped with a high-quality doublet lens
array. The fast refocusing algorithm is based on
synthetic-aperture particle image velocimetry (SAPIV). The
reconstruction quality is tested via ray-tracing of
synthetically generated particle fields. The introduced
single-camera SAPIV is applied to a convective flow within a
measurement volume of 30 x 30 x 50 mm³.
BibTeX:
@article{Skupsch2013-cq,
  author = {Skupsch, Christoph and Brücker, Christoph},
  title = {Multiple-plane particle image velocimetry using a light-field
camera}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {2}, pages = {1726--1740}, url = {http://dx.doi.org/10.1364/OE.21.001726}, doi = {http://dx.doi.org/10.1364/OE.21.001726} }
Spille, J.-H. Three-dimensional single particle tracking in a light sheet
microscope
2014 School: Universitäts-und Landesbibliothek Bonn  phdthesis URL 
Abstract: Technical development in microscopy , and particularly in
fluorescence microscopy , has facilitated the investigation of
ever smaller details in biological specimen. The combination of
specific labeling of molecular compounds, sophisticated optical
setups ...
BibTeX:
@phdthesis{Spille2014-hn,
  author = {Spille, Jan-Hendrik},
  title = {Three-dimensional single particle tracking in a light sheet
microscope}, publisher = {hss.ulb.uni-bonn.de}, school = {Universitäts-und Landesbibliothek Bonn}, year = {2014}, url = {http://hss.ulb.uni-bonn.de/2014/3589/3589.pdf} }
Spille, J.-H., Kaminski, T., Königshoven, H.-P. and Kubitscheck, U. Dynamic three-dimensional tracking of single fluorescent
nanoparticles deep inside living tissue
2012 Opt. Express
Vol. 20(18), pp. 19697-19707 
article DOI URL 
Abstract: Three-dimensional (3D) spatial information can be encoded in
two-dimensional images of fluorescent nanoparticles by
astigmatic imaging. We combined this method with light sheet
microscopy for high contrast single particle imaging up to 200
µm deep within living tissue and real-time image analysis to
determine 3D particle localizations with nanometer precision
and millisecond temporal resolution. Axial information was
instantly directed to the sample stage to keep a moving
particle within the focal plane in an active feedback loop. We
demonstrated 3D tracking of nanoparticles at an unprecedented
depth throughout large cell nuclei over several thousand
frames and a range of more than 10 µm in each spatial
dimension, while simultaneously acquiring optically sectioned
wide field images. We conclude that this 3D particle tracking
technique employing light sheet microscopy presents a valuable
extension to the nanoscopy toolbox.
BibTeX:
@article{Spille2012-yv,
  author = {Spille, Jan-Hendrik and Kaminski, Tim and Königshoven,
Heinz-Peter and Kubitscheck, Ulrich}, title = {Dynamic three-dimensional tracking of single fluorescent
nanoparticles deep inside living tissue}, journal = {Opt. Express}, publisher = {opticsinfobase.org}, year = {2012}, volume = {20}, number = {18}, pages = {19697--19707}, url = {http://dx.doi.org/10.1364/OE.20.019697}, doi = {http://dx.doi.org/10.1364/OE.20.019697} }
Stegmaier, J., Otte, J.C., Kobitski, A., Bartschat, A., Garcia, A., Nienhaus, G.U., Strähle, U. and Mikut, R. Fast segmentation of stained nuclei in terabyte-scale, time
resolved 3D microscopy image stacks
2014 PLoS One
Vol. 9(2), pp. e90036 
article DOI URL 
Abstract: Automated analysis of multi-dimensional microscopy images has
become an integral part of modern research in life science.
Most available algorithms that provide sufficient segmentation
quality, however, are infeasible for a large amount of data
due to their high complexity. In this contribution we present
a fast parallelized segmentation method that is especially
suited for the extraction of stained nuclei from microscopy
images, e.g., of developing zebrafish embryos. The idea is to
transform the input image based on gradient and normal
directions in the proximity of detected seed points such that
it can be handled by straightforward global thresholding like
Otsu's method. We evaluate the quality of the obtained
segmentation results on a set of real and simulated benchmark
images in 2D and 3D and show the algorithm's superior
performance compared to other state-of-the-art algorithms. We
achieve an up to ten-fold decrease in processing times,
allowing us to process large data sets while still providing
reasonable segmentation results.
BibTeX:
@article{Stegmaier2014-va,
  author = {Stegmaier, Johannes and Otte, Jens C and Kobitski, Andrei and
Bartschat, Andreas and Garcia, Ariel and Nienhaus, G Ulrich
and Strähle, Uwe and Mikut, Ralf}, title = {Fast segmentation of stained nuclei in terabyte-scale, time
resolved 3D microscopy image stacks}, journal = {PLoS One}, year = {2014}, volume = {9}, number = {2}, pages = {e90036}, url = {http://dx.doi.org/10.1371/journal.pone.0090036}, doi = {http://dx.doi.org/10.1371/journal.pone.0090036} }
Stelzer, E.H.K. Light-sheet fluorescence microscopy for quantitative biology 2015 Nat. Methods
Vol. 12(1), pp. 23-26 
article DOI URL 
BibTeX:
@article{Stelzer2015-pw,
  author = {Stelzer, Ernst H K},
  title = {Light-sheet fluorescence microscopy for quantitative biology},
  journal = {Nat. Methods},
  year = {2015},
  volume = {12},
  number = {1},
  pages = {23--26},
  url = {http://dx.doi.org/10.1038/nmeth.3219},
  doi = {http://dx.doi.org/10.1038/nmeth.3219}
}
Stelzer, E.H.K. S09-02 Light sheet based fluorescence microscopes (LSFM,
SPIM, DSLM) reduce phototoxic effects by several orders of
magnitude
2009 Mech. Dev.
Vol. 126, Supplement(0), pp. S36 
article DOI URL 
BibTeX:
@article{Stelzer2009-av,
  author = {Stelzer, Ernst H K},
  title = {S09-02 Light sheet based fluorescence microscopes (LSFM,
SPIM, DSLM) reduce phototoxic effects by several orders of
magnitude}, journal = {Mech. Dev.}, year = {2009}, volume = {126, Supplement}, number = {0}, pages = {S36}, url = {http://www.sciencedirect.com/science/article/pii/S0925477309011174}, doi = {http://dx.doi.org/10.1016/j.mod.2009.06.1034} }
Strobl, F., Schmitz, A. and Stelzer, E.H.K. Live imaging of Tribolium castaneum embryonic development
using light-sheet-based fluorescence microscopy
2015 Nat. Protoc.
Vol. 10(10), pp. 1486-1507 
article DOI URL 
Abstract: Tribolium castaneum has become an important insect model
organism for evolutionary developmental biology, genetics and
biotechnology. However, few protocols for live fluorescence
imaging of Tribolium have been reported, and little image data
is available. Here we provide a protocol for recording the
development of Tribolium embryos with light-sheet-based
fluorescence microscopy. The protocol can be completed in 4-7
d and provides procedural details for: embryo collection,
microscope configuration, embryo preparation and mounting,
noninvasive live imaging for up to 120 h along multiple
directions, retrieval of the live embryo once imaging is
completed, and image data processing, for which exemplary data
is provided. Stringent quality control criteria for
developmental biology studies are also discussed.
Light-sheet-based fluorescence microscopy complements existing
toolkits used to study Tribolium development, can be adapted
to other insect species, and requires no advanced imaging or
sample preparation skills.
BibTeX:
@article{Strobl2015-sh,
  author = {Strobl, Frederic and Schmitz, Alexander and Stelzer, Ernst H K},
  title = {Live imaging of Tribolium castaneum embryonic development
using light-sheet-based fluorescence microscopy}, journal = {Nat. Protoc.}, year = {2015}, volume = {10}, number = {10}, pages = {1486--1507}, url = {http://dx.doi.org/10.1038/nprot.2015.093}, doi = {http://dx.doi.org/10.1038/nprot.2015.093} }
Strobl, F. and Stelzer, E.H.K. Non-invasive long-term fluorescence live imaging of Tribolium
castaneum embryos
2014 Development
Vol. 141(11), pp. 2331-2338 
article DOI URL 
Abstract: Insect development has contributed significantly to our
understanding of metazoan development. However, most
information has been obtained by analyzing a single species,
the fruit fly Drosophila melanogaster. Embryonic development
of the red flour beetle Tribolium castaneum differs
fundamentally from that of Drosophila in aspects such as
short-germ development, embryonic leg development, extensive
extra-embryonic membrane formation and non-involuted head
development. Although Tribolium has become the second most
important insect model organism, previous live imaging
attempts have addressed only specific questions and no
long-term live imaging data of Tribolium embryogenesis have
been available. By combining light sheet-based fluorescence
microscopy with a novel mounting method, we achieved complete,
continuous and non-invasive fluorescence live imaging of
Tribolium embryogenesis at high spatiotemporal resolution. The
embryos survived the 2-day or longer imaging process,
developed into adults and produced fertile progeny. Our data
document all morphogenetic processes from the rearrangement of
the uniform blastoderm to the onset of regular muscular
movement in the same embryo and in four orientations,
contributing significantly to the understanding of Tribolium
development. Furthermore, we created a comprehensive
chronological table of Tribolium embryogenesis, integrating
most previous work and providing a reference for future
studies. Based on our observations, we provide evidence that
serosa window closure and serosa opening, although deferred by
more than 1 day, are linked. All our long-term imaging
datasets are available as a resource for the community.
Tribolium is only the second insect species, after Drosophila,
for which non-invasive long-term fluorescence live imaging has
been achieved.
BibTeX:
@article{Strobl2014-ap,
  author = {Strobl, Frederic and Stelzer, Ernst H K},
  title = {Non-invasive long-term fluorescence live imaging of Tribolium
castaneum embryos}, journal = {Development}, year = {2014}, volume = {141}, number = {11}, pages = {2331--2338}, url = {http://dx.doi.org/10.1242/dev.108795}, doi = {http://dx.doi.org/10.1242/dev.108795} }
Susaki, E.A., Tainaka, K., Perrin, D., Kishino, F., Tawara, T., Watanabe, T.M., Yokoyama, C., Onoe, H., Eguchi, M., Yamaguchi, S., Abe, T., Kiyonari, H., Shimizu, Y., Miyawaki, A., Yokota, H. and Ueda, H.R. Whole-brain imaging with single-cell resolution using chemical
cocktails and computational analysis
2014 Cell
Vol. 157(3), pp. 726-739 
article DOI URL 
Abstract: Systems-level identification and analysis of cellular circuits
in the brain will require the development of whole-brain
imaging with single-cell resolution. To this end, we performed
comprehensive chemical screening to develop a whole-brain
clearing and imaging method, termed CUBIC (clear, unobstructed
brain imaging cocktails and computational analysis). CUBIC is
a simple and efficient method involving the immersion of brain
samples in chemical mixtures containing aminoalcohols, which
enables rapid whole-brain imaging with single-photon
excitation microscopy. CUBIC is applicable to multicolor
imaging of fluorescent proteins or immunostained samples in
adult brains and is scalable from a primate brain to
subcellular structures. We also developed a whole-brain
cell-nuclear counterstaining protocol and a computational
image analysis pipeline that, together with CUBIC reagents,
enable the visualization and quantification of neural
activities induced by environmental stimulation. CUBIC enables
time-course expression profiling of whole adult brains with
single-cell resolution.
BibTeX:
@article{Susaki2014-uo,
  author = {Susaki, Etsuo A and Tainaka, Kazuki and Perrin, Dimitri and
Kishino, Fumiaki and Tawara, Takehiro and Watanabe, Tomonobu M
and Yokoyama, Chihiro and Onoe, Hirotaka and Eguchi, Megumi
and Yamaguchi, Shun and Abe, Takaya and Kiyonari, Hiroshi and
Shimizu, Yoshihiro and Miyawaki, Atsushi and Yokota, Hideo and
Ueda, Hiroki R}, title = {Whole-brain imaging with single-cell resolution using chemical
cocktails and computational analysis}, journal = {Cell}, year = {2014}, volume = {157}, number = {3}, pages = {726--739}, url = {http://dx.doi.org/10.1016/j.cell.2014.03.042}, doi = {http://dx.doi.org/10.1016/j.cell.2014.03.042} }
Swoger, J., Huisken, J. and Stelzer, E.H.K. Multiple imaging axis microscopy improves resolution for
thick-sample applications
2003 Opt. Lett.
Vol. 28(18), pp. 1654-1656 
article URL 
Abstract: The multiple imaging axis microscope (MIAM) is a wide-field
optical microscope that observes a sample simultaneously from
multiple directions without requiring the sample to be rotated
or tilted. The prototype is capable of high-resolution imaging
of the interior of a 300-microm-diameter sample consisting of
fluorescent microbeads suspended in an agarose gel. Compared
with a single-axis system, the MIAM can achieve a reduction of
the axial point-spread function elongation by a factor of 5.8
and a 3.5-fold improvement in volume resolution by simple
linear image combination techniques.
BibTeX:
@article{Swoger2003-zk,
  author = {Swoger, Jim and Huisken, Jan and Stelzer, Ernst H K},
  title = {Multiple imaging axis microscopy improves resolution for
thick-sample applications}, journal = {Opt. Lett.}, year = {2003}, volume = {28}, number = {18}, pages = {1654--1656}, url = {http://www.ncbi.nlm.nih.gov/pubmed/13677526} }
Swoger, J., Muzzopappa, M., López-Schier, H. and Sharpe, J. 4D retrospective lineage tracing using SPIM for zebrafish
organogenesis studies
2011 J. Biophotonics
Vol. 4(1-2), pp. 122-134 
article DOI URL 
Abstract: A study demonstrating an imaging framework that permits the
determination of cell lineages during organogenesis of the
posterior lateral line in zebrafish is presented. The combination
of Selective Plane Illumination Microscopy and specific
fluorescent markers allows retrospective tracking of hair cell
progenitors, and hence the derivation of their lineages within
the primodium. It is shown that, because of its superior
signal-to-noise ratio and lower photo-damaged properties, SPIM
can provide significantly higher-quality images than Spinning
Disk Confocal technology. This allows accurate 4D lineage tracing
for the hair cells over tens of hours of primordium migration and
neuromast development.
BibTeX:
@article{Swoger2011-rn,
  author = {Swoger, Jim and Muzzopappa, Mariana and López-Schier,
Hernán and Sharpe, James}, title = {4D retrospective lineage tracing using SPIM for zebrafish
organogenesis studies}, journal = {J. Biophotonics}, year = {2011}, volume = {4}, number = {1-2}, pages = {122--134}, url = {http://dx.doi.org/10.1002/jbio.201000087}, doi = {http://dx.doi.org/10.1002/jbio.201000087} }
Swoger, J., Pampaloni, F. and Stelzer, E.H.K. Imaging MDCK cysts with a single (selective) plane illumination
microscope
2014 Cold Spring Harb. Protoc.
Vol. 2014(1), pp. 114-118 
article DOI URL 
Abstract: In modern biology, most optical imaging technologies are applied
to two-dimensional cell culture systems. However, investigation
of physiological context requires specimens that display the
complex three-dimensional (3D) relationship of cells that occurs
in tissue sections and in naturally developing organisms. The
imaging of highly scattering multicellular specimens presents a
number of challenges, including limited optical penetration
depth, phototoxicity, and fluorophore bleaching.
Light-sheet-based fluorescence microscopy (LSFM) overcomes many
drawbacks of conventional fluorescence microscopy by using an
orthogonal/azimuthal fluorescence arrangement with independent
sets of lenses for illumination and detection. The specimen is
illuminated from the side with a thin light sheet that overlaps
with the focal plane of a wide-field fluorescence microscope.
Optical sectioning and minimal phototoxic damage or
photobleaching outside a small volume close to the focal plane
are intrinsic properties of LSFM. The principles of LSFM are
implemented in the single (or selective) plane illumination
microscope (SPIM). Madin-Darby canine kidney (MDCK) cysts grown
in extracellular matrix (ECM) hydrogels provide a useful model
system for studies of 3D cell biology. Here, we describe
protocols for growing MDCK cysts within 3D type I collagen or
reconstituted basement membrane (Matrigel) and for imaging these
cysts by SPIM.
BibTeX:
@article{Swoger2014-mf,
  author = {Swoger, Jim and Pampaloni, Francesco and Stelzer, Ernst H K},
  title = {Imaging MDCK cysts with a single (selective) plane illumination
microscope}, journal = {Cold Spring Harb. Protoc.}, year = {2014}, volume = {2014}, number = {1}, pages = {114--118}, url = {http://dx.doi.org/10.1101/pdb.prot080184}, doi = {http://dx.doi.org/10.1101/pdb.prot080184} }
Swoger, J., Pampaloni, F. and Stelzer, E.H.K. Imaging cellular spheroids with a single (selective) plane
illumination microscope
2014 Cold Spring Harb. Protoc.
Vol. 2014(1), pp. 106-113 
article DOI URL 
Abstract: In modern biology, most optical imaging technologies are applied
to two-dimensional cell culture systems. However, investigation
of physiological context requires specimens that display the
complex three-dimensional (3D) relationship of cells that occurs
in tissue sections and in naturally developing organisms. The
imaging of highly scattering multicellular specimens presents a
number of challenges, including limited optical penetration
depth, phototoxicity, and fluorophore bleaching.
Light-sheet-based fluorescence microscopy (LSFM) overcomes many
drawbacks of conventional fluorescence microscopy by using an
orthogonal/azimuthal fluorescence arrangement with independent
sets of lenses for illumination and detection. The specimen is
illuminated from the side with a thin light sheet that overlaps
with the focal plane of a wide-field fluorescence microscope.
Optical sectioning and minimal phototoxic damage or
photobleaching outside a small volume close to the focal plane
are intrinsic properties of LSFM. The principles of LSFM are
implemented in the single (or selective) plane illumination
microscope (SPIM). Cellular spheroids are spherical aggregations
of hundreds to thousands of cells and they provide a useful model
system for studies of 3D cell biology. Here we describe a
protocol for imaging cellular spheroids by SPIM.
BibTeX:
@article{Swoger2014-sy,
  author = {Swoger, Jim and Pampaloni, Francesco and Stelzer, Ernst H K},
  title = {Imaging cellular spheroids with a single (selective) plane
illumination microscope}, journal = {Cold Spring Harb. Protoc.}, year = {2014}, volume = {2014}, number = {1}, pages = {106--113}, url = {http://dx.doi.org/10.1101/pdb.prot080176}, doi = {http://dx.doi.org/10.1101/pdb.prot080176} }
Swoger, J., Pampaloni, F. and Stelzer, E.H.K. Light-sheet-based fluorescence microscopy for three-dimensional
imaging of biological samples
2014 Cold Spring Harb. Protoc.
Vol. 2014(1), pp. 1-8 
article DOI URL 
Abstract: In modern biology, most optical imaging technologies are applied
to two-dimensional cell culture systems; that is, they are used
in a cellular context that is defined by hard and flat surfaces.
However, a physiological context is not found in single cells
cultivated on coverslips. It requires the complex
three-dimensional (3D) relationship of cells cultivated in
extracellular matrix (ECM) gels, tissue sections, or in naturally
developing organisms. In fact, the number of applications of 3D
cell cultures in basic research as well as in drug discovery and
toxicity testing has been increasing over the past few years.
Unfortunately, the imaging of highly scattering multicellular
specimens is still challenging. The main issues are the limited
optical penetration depth, the phototoxicity, and the fluorophore
bleaching. Light-sheet-based fluorescence microscopy (LSFM)
overcomes many drawbacks of conventional fluorescence microscopy
by using an orthogonal/azimuthal fluorescence arrangement with
independent sets of lenses for illumination and detection. The
basic idea is to illuminate the specimen from the side with a
thin light sheet that overlaps with the focal plane of a
wide-field fluorescence microscope. Optical sectioning and
minimal phototoxic damage or photobleaching outside a small
volume close to the focal plane are intrinsic properties of LSFM.
We discuss the basic principles of LSFM and methods for the
preparation, embedding, and imaging of 3D specimens used in the
life sciences in an implementation of LSFM known as the single
(or selective) plane illumination microscope (SPIM).
BibTeX:
@article{Swoger2014-uv,
  author = {Swoger, Jim and Pampaloni, Francesco and Stelzer, Ernst H K},
  title = {Light-sheet-based fluorescence microscopy for three-dimensional
imaging of biological samples}, journal = {Cold Spring Harb. Protoc.}, year = {2014}, volume = {2014}, number = {1}, pages = {1--8}, url = {http://dx.doi.org/10.1101/pdb.top080168}, doi = {http://dx.doi.org/10.1101/pdb.top080168} }
Swoger, J., Verveer, P., Greger, K., Huisken, J. and Stelzer, E.H.K. Multi-view image fusion improves resolution in
three-dimensional microscopy
2007 Opt. Express
Vol. 15(13), pp. 8029-8042 
article URL 
Abstract: A non-blind, shift-invariant image processing technique that
fuses multi-view three-dimensional image data sets into a
single, high quality three-dimensional image is presented. It
is effective for 1) improving the resolution and isotropy in
images of transparent specimens, and 2) improving the
uniformity of the image quality of partially opaque samples.
This is demonstrated with fluorescent samples such as
Drosophila melanogaster and Medaka embryos and pollen grains
imaged by Selective Plane Illumination Microscopy (SPIM). The
application of the algorithm to SPIM data yields
high-resolution images of organ structure and gene expression,
in some cases at a sub-cellular level, throughout specimens
ranging from several microns up to a millimeter in size.
BibTeX:
@article{Swoger2007-lx,
  author = {Swoger, Jim and Verveer, Peter and Greger, Klaus and Huisken,
Jan and Stelzer, Ernst H K}, title = {Multi-view image fusion improves resolution in
three-dimensional microscopy}, journal = {Opt. Express}, year = {2007}, volume = {15}, number = {13}, pages = {8029--8042}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19547131} }
Takao, D., Taniguchi, A., Takeda, T., Sonobe, S. and Nonaka, S. High-speed imaging of amoeboid movements using light-sheet
microscopy
2012 PLoS One
Vol. 7(12), pp. e50846 
article DOI URL 
Abstract: Light-sheet microscopy has been developed as a powerful tool
for live imaging in biological studies. The efficient
illumination of specimens using light-sheet microscopy makes
it highly amenable to high-speed imaging. We therefore applied
this technology to the observation of amoeboid movements,
which are too rapid to capture with conventional microscopy.
To simplify the setup of the optical system, we utilized the
illumination optics from a conventional confocal laser
scanning microscope. Using this set-up we achieved high-speed
imaging of amoeboid movements. Three-dimensional images were
captured at the recording rate of 40 frames/s and clearly
outlined the fine structures of fluorescent-labeled amoeboid
cellular membranes. The quality of images obtained by our
system was sufficient for subsequent quantitative analysis for
dynamics of amoeboid movements. This study demonstrates the
application of light-sheet microscopy for high-speed imaging
of biological specimens.
BibTeX:
@article{Takao2012-uu,
  author = {Takao, Daisuke and Taniguchi, Atsushi and Takeda, Takaaki and
Sonobe, Seiji and Nonaka, Shigenori}, title = {High-speed imaging of amoeboid movements using light-sheet
microscopy}, journal = {PLoS One}, year = {2012}, volume = {7}, number = {12}, pages = {e50846}, url = {http://dx.doi.org/10.1371/journal.pone.0050846}, doi = {http://dx.doi.org/10.1371/journal.pone.0050846} }
Taormina, M.J., Jemielita, M., Stephens, W.Z., Burns, A.R., Troll, J.V., Parthasarathy, R. and Guillemin, K. Investigating bacterial-animal symbioses with light sheet
microscopy
2012 Biol. Bull.
Vol. 223(1), pp. 7-20 
article URL 
Abstract: Microbial colonization of the digestive tract is a crucial
event in vertebrate development, required for maturation of
host immunity and establishment of normal digestive
physiology. Advances in genomic, proteomic, and metabolomic
technologies are providing a more detailed picture of the
constituents of the intestinal habitat, but these approaches
lack the spatial and temporal resolution needed to
characterize the assembly and dynamics of microbial
communities in this complex environment. We report the use of
light sheet microscopy to provide high-resolution imaging of
bacterial colonization of the intestine of Danio rerio, the
zebrafish. The method allows us to characterize bacterial
population dynamics across the entire organ and the behaviors
of individual bacterial and host cells throughout the
colonization process. The large four-dimensional data sets
generated by these imaging approaches require new strategies
for image analysis. When integrated with other ``omics'' data
sets, information about the spatial and temporal dynamics of
microbial cells within the vertebrate intestine will provide
new mechanistic insights into how microbial communities
assemble and function within hosts.
BibTeX:
@article{Taormina2012-gg,
  author = {Taormina, Michael J and Jemielita, Matthew and Stephens, W Zac
and Burns, Adam R and Troll, Joshua V and Parthasarathy,
Raghuveer and Guillemin, Karen}, title = {Investigating bacterial-animal symbioses with light sheet
microscopy}, journal = {Biol. Bull.}, year = {2012}, volume = {223}, number = {1}, pages = {7--20}, url = {http://www.ncbi.nlm.nih.gov/pubmed/22983029} }
Taxis, C., Maeder, C., Reber, S., Rathfelder, N., Miura, K., Greger, K., Stelzer, E.H.K. and Knop, M. Dynamic organization of the actin cytoskeleton during meiosis
and spore formation in budding yeast
2006 Traffic
Vol. 7(12), pp. 1628-1642 
article DOI URL 
Abstract: During sporulation in Saccharomyces cerevisiae, the four
daughter cells (spores) are formed inside the boundaries of
the mother cell. Here, we investigated the dynamics of spore
assembly and the actin cytoskeleton during this process, as
well as the requirements for filamentous actin during the
different steps of spore formation. We found no evidence for a
polarized actin cytoskeleton during sporulation. Instead, a
highly dynamic network of non-polarized actin cables is
present underneath the plasma membrane of the mother cell. We
found that a fraction of prospore membrane (PSM) precursors
are transported along the actin cables. The velocity of PSM
precursors is diminished if Myo2p or Tpm1/2p function is
impaired. Filamentous actin is not essential for meiotic
progression, for shaping of the PSMs or for post-meiotic
cytokinesis. However, actin is essential for spore wall
formation. This requires the function of the Arp2/3p complex
and involves large carbohydrate-rich compartments, which may
be chitosome analogous structures.
BibTeX:
@article{Taxis2006-wj,
  author = {Taxis, Christof and Maeder, Celine and Reber, Simone and
Rathfelder, Nicole and Miura, Kota and Greger, Klaus and
Stelzer, Ernst H K and Knop, Michael}, title = {Dynamic organization of the actin cytoskeleton during meiosis
and spore formation in budding yeast}, journal = {Traffic}, year = {2006}, volume = {7}, number = {12}, pages = {1628--1642}, url = {http://dx.doi.org/10.1111/j.1600-0854.2006.00496.x}, doi = {http://dx.doi.org/10.1111/j.1600-0854.2006.00496.x} }
Taylor, J.M. Optically gated beating-heart imaging 2014 Front. Physiol.
Vol. 5, pp. 481 
article DOI URL 
Abstract: The constant motion of the beating heart presents an obstacle
to clear optical imaging, especially 3D imaging, in small
animals where direct optical imaging would otherwise be
possible. Gating techniques exploit the periodic motion of the
heart to computationally ``freeze'' this movement and overcome
motion artifacts. Optically gated imaging represents a recent
development of this, where image analysis is used to
synchronize acquisition with the heartbeat in a completely
non-invasive manner. This article will explain the concept of
optical gating, discuss a range of different implementation
strategies and their strengths and weaknesses. Finally we will
illustrate the usefulness of the technique by discussing
applications where optical gating has facilitated novel
biological findings by allowing 3D in vivo imaging of cardiac
myocytes in their natural environment of the beating heart.
BibTeX:
@article{Taylor2014-fn,
  author = {Taylor, Jonathan M},
  title = {Optically gated beating-heart imaging},
  journal = {Front. Physiol.},
  year = {2014},
  volume = {5},
  pages = {481},
  url = {http://dx.doi.org/10.3389/fphys.2014.00481},
  doi = {http://dx.doi.org/10.3389/fphys.2014.00481}
}
Taylor, J.M., Saunter, C.D., Bourgenot, C., Girkin, J.M. and Love, G.D. Realtime wavefront sensing in a SPIM microscope, and active
aberration tracking
2015 SPIE BiOS, pp. 93350A-93350A-6  inproceedings DOI URL 
BibTeX:
@inproceedings{Taylor2015-wd,
  author = {Taylor, Jonathan M and Saunter, Christopher D and Bourgenot,
Cyril and Girkin, John M and Love, Gordon D}, title = {Realtime wavefront sensing in a SPIM microscope, and active
aberration tracking}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {93350A--93350A--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2214150}, doi = {http://dx.doi.org/10.1117/12.2080061} }
Taylor, J.M., Saunter, C.D., Love, G.D. and Girkin, J.M. High-resolution time-resolved 3D optical microscopy inside the
beating zebrafish heart using prospective optical gating
2013 SPIE BiOS, pp. 859302-859302-8  inproceedings DOI URL 
BibTeX:
@inproceedings{Taylor2013-tj,
  author = {Taylor, J M and Saunter, C D and Love, G D and Girkin, J M},
  title = {High-resolution time-resolved 3D optical microscopy inside the
beating zebrafish heart using prospective optical gating}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2013}, pages = {859302--859302--8}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1672724}, doi = {http://dx.doi.org/10.1117/12.2005886} }
Taylor, J.M., Saunter, C.D., Love, G.D. and Girkin, J.M. Prospective gating for 3D imaging of the beating zebrafish
heart in embryonic development studies
2012 SPIE BiOS, pp. 822716-822716-7  inproceedings DOI URL 
Abstract: ... We present results obtained using our SPIM system including
3D reconstructions of the living, beating heart , acquired using
optical gating without the ... Keywords: Optical Microscopy,
Selective Plane Illumination, Synchronization , Fluorescence
imaging 1. INTRODUCTION ...
BibTeX:
@inproceedings{Taylor2012-go,
  author = {Taylor, J M and Saunter, C D and Love, G D and Girkin, J M},
  title = {Prospective gating for 3D imaging of the beating zebrafish
heart in embryonic development studies}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2012}, pages = {822716--822716--7}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1276546}, doi = {http://dx.doi.org/10.1117/12.908164} }
Taylor, J.M., Saunter, C.D., Love, G.D., Girkin, J.M., Henderson, D.J. and Chaudhry, B. Real-time optical gating for three-dimensional beating heart
imaging
2011 J. Biomed. Opt.
Vol. 16(11), pp. 116021 
article DOI URL 
Abstract: We demonstrate real-time microscope image gating to an
arbitrary position in the cycle of the beating heart of a
zebrafish embryo. We show how this can be used for
high-precision prospective gating of fluorescence image slices
of the moving heart. We also present initial results
demonstrating the application of this technique to 3-D
structural imaging of the beating embryonic heart.
BibTeX:
@article{Taylor2011-dy,
  author = {Taylor, Jonathan M and Saunter, Christopher D and Love, Gordon
D and Girkin, John M and Henderson, Deborah J and Chaudhry,
Bill}, title = {Real-time optical gating for three-dimensional beating heart
imaging}, journal = {J. Biomed. Opt.}, year = {2011}, volume = {16}, number = {11}, pages = {116021}, url = {http://dx.doi.org/10.1117/1.3652892}, doi = {http://dx.doi.org/10.1117/1.3652892} }
Temerinac-Ott, M., Ronneberger, O., Nitschke, R., Driever, W. and Burkhardt, H. Spatially-variant Lucy-Richardson deconvolution for multiview
fusion of microscopical 3D images
2011 Biomedical Imaging: From Nano to Macro, 2011 IEEE
International Symposium on, pp. 899-904 
inproceedings DOI URL 
Abstract: A framework for fast multiview fusion of Single Plane
Illumination Microscopy (SPIM) images based on a
spatially-variant point spread function (PSF) model is
presented. For the multiview fusion a new algorithm based on the
regularized Lucy-Richardson deconvolution and the Overlap-Save
method is developed and tested on SPIM images. In the algorithm
the image is decomposed into small blocks which are processed
separately thus saving memory space and allowing for parallel
processing.
BibTeX:
@inproceedings{Temerinac-Ott2011-gr,
  author = {Temerinac-Ott, M and Ronneberger, O and Nitschke, R and Driever,
W and Burkhardt, H}, title = {Spatially-variant Lucy-Richardson deconvolution for multiview
fusion of microscopical 3D images}, booktitle = {Biomedical Imaging: From Nano to Macro, 2011 IEEE
International Symposium on}, year = {2011}, pages = {899--904}, url = {http://dx.doi.org/10.1109/ISBI.2011.5872549}, doi = {http://dx.doi.org/10.1109/ISBI.2011.5872549} }
Temerinac-Ott, M., Ronneberger, O., Ochs, P., Driever, W., Brox, T. and Burkhardt, H. Multiview deblurring for 3-D images from light-sheet-based
fluorescence microscopy
2012 IEEE Trans. Image Process.
Vol. 21(4), pp. 1863-1873 
article DOI URL 
Abstract: We propose an algorithm for 3-D multiview deblurring using
spatially variant point spread functions (PSFs). The algorithm
is applied to multiview reconstruction of volumetric
microscopy images. It includes registration and estimation of
the PSFs using irregularly placed point markers (beads). We
formulate multiview deblurring as an energy minimization
problem subject to L1-regularization. Optimization is based on
the regularized Lucy-Richardson algorithm, which we extend to
deal with our more general model. The model parameters are
chosen in a profound way by optimizing them on a realistic
training set. We quantitatively and qualitatively compare with
existing methods and show that our method provides better
signal-to-noise ratio and increases the resolution of the
reconstructed images.
BibTeX:
@article{Temerinac-Ott2012-jy,
  author = {Temerinac-Ott, Maja and Ronneberger, Olaf and Ochs, Peter and
Driever, Wolfgang and Brox, Thomas and Burkhardt, Hans}, title = {Multiview deblurring for 3-D images from light-sheet-based
fluorescence microscopy}, journal = {IEEE Trans. Image Process.}, year = {2012}, volume = {21}, number = {4}, pages = {1863--1873}, url = {http://dx.doi.org/10.1109/TIP.2011.2181528}, doi = {http://dx.doi.org/10.1109/TIP.2011.2181528} }
Tokunaga, M., Imamoto, N. and Sakata-Sogawa, K. Highly inclined thin illumination enables clear
single-molecule imaging in cells
2008 Nat. Methods
Vol. 5(2), pp. 159-161 
article DOI URL 
Abstract: We describe a simple illumination method of fluorescence
microscopy for molecular imaging. Illumination by a highly
inclined and thin beam increases image intensity and decreases
background intensity, yielding a signal/background ratio about
eightfold greater than that of epi-illumination. A high ratio
yielded clear single-molecule images and three-dimensional
images using cultured mammalian cells, enabling one to
visualize and quantify molecular dynamics, interactions and
kinetics in cells for molecular systems biology.
BibTeX:
@article{Tokunaga2008-rd,
  author = {Tokunaga, Makio and Imamoto, Naoko and Sakata-Sogawa, Kumiko},
  title = {Highly inclined thin illumination enables clear
single-molecule imaging in cells}, journal = {Nat. Methods}, year = {2008}, volume = {5}, number = {2}, pages = {159--161}, url = {http://dx.doi.org/10.1038/nmeth1171}, doi = {http://dx.doi.org/10.1038/nmeth1171} }
Tomer, R., Khairy, K., Amat, F. and Keller, P.J. Quantitative high-speed imaging of entire developing embryos
with simultaneous multiview light-sheet microscopy
2012 Nat. Methods
Vol. 9(7), pp. 755-763 
article DOI URL 
Abstract: Live imaging of large biological specimens is fundamentally
limited by the short optical penetration depth of light
microscopes. To maximize physical coverage, we developed the
SiMView technology framework for high-speed in vivo imaging,
which records multiple views of the specimen simultaneously.
SiMView consists of a light-sheet microscope with four
synchronized optical arms, real-time electronics for long-term
sCMOS-based image acquisition at 175 million voxels per
second, and computational modules for high-throughput image
registration, segmentation, tracking and real-time management
of the terabytes of multiview data recorded per specimen. We
developed one-photon and multiphoton SiMView implementations
and recorded cellular dynamics in entire Drosophila
melanogaster embryos with 30-s temporal resolution throughout
development. We furthermore performed high-resolution
long-term imaging of the developing nervous system and
followed neuroblast cell lineages in vivo. SiMView data sets
provide quantitative morphological information even for fast
global processes and enable accurate automated cell tracking
in the entire early embryo.
BibTeX:
@article{Tomer2012-ev,
  author = {Tomer, Raju and Khairy, Khaled and Amat, Fernando and Keller,
Philipp J}, title = {Quantitative high-speed imaging of entire developing embryos
with simultaneous multiview light-sheet microscopy}, journal = {Nat. Methods}, year = {2012}, volume = {9}, number = {7}, pages = {755--763}, url = {http://dx.doi.org/10.1038/nmeth.2062}, doi = {http://dx.doi.org/10.1038/nmeth.2062} }
Tomer, R., Khairy, K. and Keller, P.J. Light sheet microscopy in cell biology 2013 Methods Mol. Biol.
Vol. 931, pp. 123-137 
article DOI URL 
Abstract: Light sheet-based fluorescence microscopy (LSFM) is emerging
as a powerful imaging technique for the life sciences. LSFM
provides an exceptionally high imaging speed, high
signal-to-noise ratio, low level of photo-bleaching, and good
optical penetration depth. This unique combination of
capabilities makes light sheet-based microscopes highly
suitable for live imaging applications. Here, we provide an
overview of light sheet-based microscopy assays for in vitro
and in vivo imaging of biological samples, including cell
extracts, soft gels, and large multicellular organisms. We
furthermore describe computational tools for basic image
processing and data inspection.
BibTeX:
@article{Tomer2013-un,
  author = {Tomer, Raju and Khairy, Khaled and Keller, Philipp J},
  title = {Light sheet microscopy in cell biology},
  journal = {Methods Mol. Biol.},
  year = {2013},
  volume = {931},
  pages = {123--137},
  url = {http://dx.doi.org/10.1007/978-1-62703-056-4_7},
  doi = {http://dx.doi.org/10.1007/978-1-62703-056-4\_7}
}
Tomer, R., Khairy, K. and Keller, P.J. Shedding light on the system: studying embryonic development
with light sheet microscopy
2011 Curr. Opin. Genet. Dev.
Vol. 21(5), pp. 558-565 
article DOI URL 
Abstract: Light sheet-based fluorescence microscopy (LSFM) is emerging
as a powerful imaging technique for the life sciences. LSFM
provides an exceptionally high imaging speed, high
signal-to-noise ratio, low level of photo-bleaching and good
optical penetration depth. This unique combination of
capabilities makes light sheet-based microscopes highly
suitable for live imaging applications. There is an
outstanding potential in applying this technology to the
quantitative study of embryonic development. Here, we provide
an overview of the different basic implementations of LSFM,
review recent technical advances in the field and highlight
applications in the context of embryonic development. We
conclude with a discussion of promising future directions.
BibTeX:
@article{Tomer2011-wv,
  author = {Tomer, Raju and Khairy, Khaled and Keller, Philipp J},
  title = {Shedding light on the system: studying embryonic development
with light sheet microscopy}, journal = {Curr. Opin. Genet. Dev.}, publisher = {Elsevier Ltd}, year = {2011}, volume = {21}, number = {5}, pages = {558--565}, url = {http://dx.doi.org/10.1016/j.gde.2011.07.003}, doi = {http://dx.doi.org/10.1016/j.gde.2011.07.003} }
Trivedi, V., Truong, T.V., Trinh, L.A., Holland, D.B., Liebling, M. and Fraser, S.E. Dynamic structure and protein expression of the live embryonic
heart captured by 2-photon light sheet microscopy and
retrospective registration
2015 Biomed. Opt. Express
Vol. 6(6), pp. 2056-2066 
article DOI URL 
Abstract: We present an imaging and image reconstruction pipeline that
captures the dynamic three-dimensional beating motion of the
live embryonic zebrafish heart at subcellular resolution.
Live, intact zebrafish embryos were imaged using 2-photon
light sheet microscopy, which offers deep and fast imaging at
70 frames per second, and the individual optical sections were
assembled into a full 4D reconstruction of the beating heart
using an optimized retrospective image registration algorithm.
This imaging and reconstruction platform permitted us to
visualize protein expression patterns at endogenous
concentrations in zebrafish gene trap lines.
BibTeX:
@article{Trivedi2015-uh,
  author = {Trivedi, Vikas and Truong, Thai V and Trinh, Le A and Holland,
Daniel B and Liebling, Michael and Fraser, Scott E}, title = {Dynamic structure and protein expression of the live embryonic
heart captured by 2-photon light sheet microscopy and
retrospective registration}, journal = {Biomed. Opt. Express}, year = {2015}, volume = {6}, number = {6}, pages = {2056--2066}, url = {http://dx.doi.org/10.1364/BOE.6.002056}, doi = {http://dx.doi.org/10.1364/BOE.6.002056} }
Truong, T.V., Supatto, W., Koos, D.S., Choi, J.M. and Fraser, S.E. Deep and fast live imaging with two-photon scanned light-sheet
microscopy
2011 Nat. Methods
Vol. 8(9), pp. 757-760 
article DOI URL 
Abstract: We implemented two-photon scanned light-sheet microscopy,
combining nonlinear excitation with orthogonal illumination of
light-sheet microscopy, and showed its excellent performance
for in vivo, cellular-resolution, three-dimensional imaging of
large biological samples. Live imaging of fruit fly and
zebrafish embryos confirmed that the technique can be used to
image up to twice deeper than with one-photon light-sheet
microscopy and more than ten times faster than with
point-scanning two-photon microscopy without compromising
normal biology.
BibTeX:
@article{Truong2011-aq,
  author = {Truong, Thai V and Supatto, Willy and Koos, David S and Choi,
John M and Fraser, Scott E}, title = {Deep and fast live imaging with two-photon scanned light-sheet
microscopy}, journal = {Nat. Methods}, year = {2011}, volume = {8}, number = {9}, pages = {757--760}, url = {http://dx.doi.org/10.1038/nmeth.1652}, doi = {http://dx.doi.org/10.1038/nmeth.1652} }
Turaga, D. and Holy, T.E. Aberrations and their correction in light-sheet microscopy: a
low-dimensional parametrization
2013 Biomed. Opt. Express
Vol. 4(9), pp. 1654-1661 
article DOI URL 
Abstract: Light sheet microscopy allows rapid imaging of
three-dimensional fluorescent samples, using illumination and
detection axes that are orthogonal. For imaging large samples,
this often forces the objective to be tilted relative to the
sample's surface; for samples that are not precisely matched
to the immersion medium index, this tilt introduces
aberrations. Here we calculate the nature of these aberrations
for a simple tissue model, and show that a low-dimensional
parametrization of these aberrations facilitates online
correction via a deformable mirror without introduction of
beads or other fiducial markers. We use this approach to
demonstrate improved image quality in living tissue.
BibTeX:
@article{Turaga2013-gg,
  author = {Turaga, Diwakar and Holy, Timothy E},
  title = {Aberrations and their correction in light-sheet microscopy: a
low-dimensional parametrization}, journal = {Biomed. Opt. Express}, year = {2013}, volume = {4}, number = {9}, pages = {1654--1661}, url = {http://dx.doi.org/10.1364/BOE.4.001654}, doi = {http://dx.doi.org/10.1364/BOE.4.001654} }
Turaga, D. and Holy, T.E. Image-based calibration of a deformable mirror in wide-field
microscopy
2010 Appl. Opt.
Vol. 49(11), pp. 2030-2040 
article DOI URL 
Abstract: Optical aberrations limit resolution in biological tissues,
and their influence is particularly large for promising
techniques such as light-sheet microscopy. In principle, image
quality might be improved by adaptive optics (AO), in which
aberrations are corrected by using a deformable mirror (DM).
To implement AO in microscopy, one requires a method to
measure wavefront aberrations, but the most commonly used
methods have limitations for samples lacking point-source
emitters. Here we implement an image-based wavefront-sensing
technique, a variant of generalized phase-diverse imaging
called multiframe blind deconvolution, and exploit it to
calibrate a DM in a light-sheet microscope. We describe two
methods of parameterizing the influence of the DM on
aberrations: a traditional Zernike expansion requiring 1040
parameters, and a direct physical model of the DM requiring
just 8 or 110 parameters. By randomizing voltages on all
actuators, we show that the Zernike expansion successfully
predicts wavefronts to an accuracy of approximately 30 nm
(rms) even for large aberrations. We thus show that
image-based wavefront sensing, which requires no additional
optical equipment, allows a simple but powerful method to
calibrate a deformable optical element in a microscope
setting.
BibTeX:
@article{Turaga2010-bc,
  author = {Turaga, Diwakar and Holy, Timothy E},
  title = {Image-based calibration of a deformable mirror in wide-field
microscopy}, journal = {Appl. Opt.}, year = {2010}, volume = {49}, number = {11}, pages = {2030--2040}, url = {http://dx.doi.org/10.1364/AO.49.002030}, doi = {http://dx.doi.org/10.1364/AO.49.002030} }
Turaga, D. and Holy, T.E. Miniaturization and defocus correction for objective-coupled
planar illumination microscopy
2008 Opt. Lett.
Vol. 33(20), pp. 2302-2304 
article URL 
Abstract: Recently, a light sheet-based technique called
objective-coupled planar illumination (OCPI) microscopy
[Holekamp et al., Neuron 57, 661 (2008)] was shown to permit
low-phototoxicity, high-speed, three-dimensional fluorescence
imaging of extended tissue samples. Here, we introduce two
major improvements in OCPI microscopy. First, we miniaturize
the objective coupler by using a uniaxial gradient-index lens
to produce the light sheet. Second, we demonstrate
theoretically and experimentally that refractive index
mismatch at the fluid/tissue interface causes a significant
defocus aberration. By introducing the ability to tune the
angle of the light sheet, we show that defocus correction in a
miniaturized OCPI microscope leads to a significant
improvement in image sharpness deeper into tissue.
BibTeX:
@article{Turaga2008-kj,
  author = {Turaga, Diwakar and Holy, Timothy E},
  title = {Miniaturization and defocus correction for objective-coupled
planar illumination microscopy}, journal = {Opt. Lett.}, year = {2008}, volume = {33}, number = {20}, pages = {2302--2304}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18923603} }
Udan, R.S., Piazza, V.G., Hsu, C.-W., Hadjantonakis, A.-K. and Dickinson, M.E. Quantitative imaging of cell dynamics in mouse embryos using
light-sheet microscopy
2014 Development
Vol. 141(22), pp. 4406-4414 
article DOI URL 
Abstract: Single/selective-plane illumination, or light-sheet, systems
offer several advantages over other fluorescence microscopy
methods for live, 3D microscopy. These systems are valuable
for studying embryonic development in several animal systems,
such as Drosophila, C. elegans and zebrafish. The geometry of
the light path in this form of microscopy requires the sample
to be accessible from multiple sides and fixed in place so
that it can be rotated around a single axis. Popular methods
for mounting include hanging the specimen from a pin or
embedding it in 1-2% agarose. These methods can be
particularly problematic for certain samples, such as
post-implantation mouse embryos, that expand significantly in
size and are very delicate and sensitive to mounting. To
overcome the current limitations and to establish a robust
strategy for long-term (24 h) time-lapse imaging of E6.5-8.5
mouse embryos with light-sheet microscopy, we developed and
tested a method using hollow agarose cylinders designed to
accommodate for embryonic growth, yet provide boundaries to
minimize tissue drift and enable imaging in multiple
orientations. Here, we report the first 24-h time-lapse
sequences of post-implantation mouse embryo development with
light-sheet microscopy. We demonstrate that light-sheet
imaging can provide both quantitative data for tracking
changes in morphogenesis and reveal new insights into mouse
embryogenesis. Although we have used this approach for imaging
mouse embryos, it can be extended to imaging other types of
embryos as well as tissue explants.
BibTeX:
@article{Udan2014-fe,
  author = {Udan, Ryan S and Piazza, Victor G and Hsu, Chih-Wei and
Hadjantonakis, Anna-Katerina and Dickinson, Mary E}, title = {Quantitative imaging of cell dynamics in mouse embryos using
light-sheet microscopy}, journal = {Development}, year = {2014}, volume = {141}, number = {22}, pages = {4406--4414}, url = {http://dx.doi.org/10.1242/dev.111021}, doi = {http://dx.doi.org/10.1242/dev.111021} }
Vermeer, J.E.M., von Wangenheim, D., Barberon, M., Lee, Y., Stelzer, E.H.K., Maizel, A. and Geldner, N. A spatial accommodation by neighboring cells is required for
organ initiation in Arabidopsis
2014 Science
Vol. 343(6167), pp. 178-183 
article DOI URL 
Abstract: Lateral root formation in plants can be studied as the process
of interaction between chemical signals and physical forces
during development. Lateral root primordia grow through
overlying cell layers that must accommodate this incursion.
Here, we analyze responses of the endodermis, the immediate
neighbor to an initiating lateral root. Endodermal cells
overlying lateral root primordia lose volume, change shape,
and relinquish their tight junction-like diffusion barrier to
make way for the emerging lateral root primordium. Endodermal
feedback is absolutely required for initiation and growth of
lateral roots, and we provide evidence that this is mediated
by controlled volume loss in the endodermis. We propose that
turgidity and rigid cell walls, typical of plants, impose
constraints that are specifically modified for a given
developmental process.
BibTeX:
@article{Vermeer2014-wz,
  author = {Vermeer, Joop E M and von Wangenheim, Daniel and Barberon,
Marie and Lee, Yuree and Stelzer, Ernst H K and Maizel, Alexis
and Geldner, Niko}, title = {A spatial accommodation by neighboring cells is required for
organ initiation in Arabidopsis}, journal = {Science}, year = {2014}, volume = {343}, number = {6167}, pages = {178--183}, url = {http://dx.doi.org/10.1126/science.1245871}, doi = {http://dx.doi.org/10.1126/science.1245871} }
Verveer, P.J., Swoger, J., Pampaloni, F., Greger, K., Marcello, M. and Stelzer, E.H.K. High-resolution three-dimensional imaging of large specimens
with light sheet-based microscopy
2007 Nat. Methods
Vol. 4(4), pp. 311-313 
article DOI URL 
Abstract: We report that single (or selective) plane illumination
microscopy (SPIM), combined with a new deconvolution
algorithm, provides a three-dimensional spatial resolution
exceeding that of confocal fluorescence microscopy in large
samples. We demonstrate this by imaging large living
multicellular specimens obtained in a three-dimensional cell
culture. The ability to rapidly image large samples at high
resolution with minimal photodamage provides new opportunities
especially for the study of subcellular processes in large
living specimens.
BibTeX:
@article{Verveer2007-am,
  author = {Verveer, Peter J and Swoger, Jim and Pampaloni, Francesco and
Greger, Klaus and Marcello, Marco and Stelzer, Ernst H K}, title = {High-resolution three-dimensional imaging of large specimens
with light sheet-based microscopy}, journal = {Nat. Methods}, year = {2007}, volume = {4}, number = {4}, pages = {311--313}, url = {http://dx.doi.org/10.1038/nmeth1017}, doi = {http://dx.doi.org/10.1038/nmeth1017} }
Vettenburg, T., Dalgarno, H.I.C., Čižmár, T., Gunn-Moore, F.J. and Dholakia, K. Increasing the resolution of light sheet microscopy in the
presence of aberrations
2013 SPIE BiOS, pp. 858912-858912-6  inproceedings DOI URL 
Abstract: abstract Single plane illumination microscopy (SPIM) allows
rapid imaging of large, three- dimensional, samples of living
tissue. The thin light sheet ensures high contrast whilst photo-
bleaching and damage are kept to a minimum. However, many
specimen of interest have ...
BibTeX:
@inproceedings{Vettenburg2013-jv,
  author = {Vettenburg, T and Dalgarno, H I C and Čižmár, T and
Gunn-Moore, F J and Dholakia, K}, title = {Increasing the resolution of light sheet microscopy in the
presence of aberrations}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2013}, pages = {858912--858912--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1656302}, doi = {http://dx.doi.org/10.1117/12.2003828} }
Vettenburg, T., Dalgarno, H.I.C., Nylk, J., Coll-Lladó, C., Ferrier, D.E.K., Čižmár, T., Gunn-Moore, F.J. and Dholakia, K. Light-sheet microscopy using an Airy beam 2014 Nat. Methods
Vol. 11(5), pp. 541-544 
article DOI URL 
Abstract: Light-sheet microscopy facilitates rapid, high-contrast,
volumetric imaging with minimal sample exposure. However, the
rapid divergence of a traditional Gaussian light sheet
restricts the field of view (FOV) that provides innate
subcellular resolution. We show that the Airy beam innately
yields high contrast and resolution up to a tenfold larger
FOV. In contrast to the Bessel beam, which also provides an
increased FOV, the Airy beam's characteristic asymmetric
excitation pattern results in all fluorescence contributing
positively to the contrast, enabling a step change for
light-sheet microscopy.
BibTeX:
@article{Vettenburg2014-rj,
  author = {Vettenburg, Tom and Dalgarno, Heather I C and Nylk, Jonathan
and Coll-Lladó, Clara and Ferrier, David E K and
Čižmár, Tomáš and Gunn-Moore, Frank J and
Dholakia, Kishan}, title = {Light-sheet microscopy using an Airy beam}, journal = {Nat. Methods}, year = {2014}, volume = {11}, number = {5}, pages = {541--544}, url = {http://dx.doi.org/10.1038/nmeth.2922}, doi = {http://dx.doi.org/10.1038/nmeth.2922} }
Vladimirov, N., Mu, Y., Kawashima, T., Bennett, D.V., Yang, C.-T., Looger, L.L., Keller, P.J., Freeman, J. and Ahrens, M.B. Light-sheet functional imaging in fictively behaving zebrafish 2014 Nat. Methods
Vol. 11(9), pp. 883-884 
article DOI URL 
BibTeX:
@article{Vladimirov2014-hv,
  author = {Vladimirov, Nikita and Mu, Yu and Kawashima, Takashi and
Bennett, Davis V and Yang, Chao-Tsung and Looger, Loren L and
Keller, Philipp J and Freeman, Jeremy and Ahrens, Misha B}, title = {Light-sheet functional imaging in fictively behaving zebrafish}, journal = {Nat. Methods}, year = {2014}, volume = {11}, number = {9}, pages = {883--884}, url = {http://dx.doi.org/10.1038/nmeth.3040}, doi = {http://dx.doi.org/10.1038/nmeth.3040} }
Voie, A.H. Imaging the intact guinea pig tympanic bulla by
orthogonal-plane fluorescence optical sectioning microscopy
2002 Hear. Res.
Vol. 171(1-2), pp. 119-128 
article URL 
Abstract: Orthogonal-plane fluorescence optical sectioning (OPFOS)
microscopy was developed for the purpose of making
quantitative measurements of the intact mammalian cochlea and
to facilitate 3D reconstructions of complex features. A new
version of this imaging apparatus was built with a specimen
chamber designed to accommodate samples as large as the intact
guinea pig bulla. This method left the cochlear connections
with the vestibular system and with the ossicles of the middle
ear undisturbed, providing views within the cochlea with no
breaches of its structural integrity. Since the features
within the bulla were not physically touched during the
preparation process, the risk of damage was minimized, and
were imaged in relatively pristine condition with spatial
resolution to 16 microm. A description of the imaging method
and specimen preparation procedure is presented, as are images
of features from the cochlea, ossicles, and vestibular system.
BibTeX:
@article{Voie2002-jm,
  author = {Voie, Arne H},
  title = {Imaging the intact guinea pig tympanic bulla by
orthogonal-plane fluorescence optical sectioning microscopy}, journal = {Hear. Res.}, year = {2002}, volume = {171}, number = {1-2}, pages = {119--128}, url = {http://www.ncbi.nlm.nih.gov/pubmed/12204356} }
Voie, A.H., Burns, D.H. and Spelman, F.A. Orthogonal-plane fluorescence optical sectioning:
three-dimensional imaging of macroscopic biological specimens
1993 J. Microsc.
Vol. 170(Pt 3), pp. 229-236 
article URL 
Abstract: An imaging technique called orthogonal-plane fluorescence
optical sectioning (OPFOS) was developed to image the internal
architecture of the cochlea. Expressions for the
three-dimensional point spread function and the axial and
lateral resolution are derived. Methodologies for tissue
preparation and for construction, alignment, calibration and
characterization of an OPFOS apparatus are presented. The
instrument described produced focused, high-resolution images
of optical sections of an intact, excised guinea-pig cochlea.
The lateral and axial resolutions of the images were 10 and 26
microns, respectively, within a 1.5-mm field of view.
BibTeX:
@article{Voie1993-gk,
  author = {Voie, A H and Burns, D H and Spelman, F A},
  title = {Orthogonal-plane fluorescence optical sectioning:
three-dimensional imaging of macroscopic biological specimens}, journal = {J. Microsc.}, year = {1993}, volume = {170}, number = {Pt 3}, pages = {229--236}, url = {http://www.ncbi.nlm.nih.gov/pubmed/8371260} }
Voie, A.H. and Spelman, F.A. Three-dimensional reconstruction of the cochlea from
two-dimensional images of optical sections
1995 Comput. Med. Imaging Graph.
Vol. 19(5), pp. 377-384 
article URL 
Abstract: This paper describes a methodology for three-dimensional (3D)
computer-aided reconstruction of the guinea pig cochlea using
orthogonal-plane fluorescence optical sectioning. Specimens
are sectioned optically, allowing them to remain intact during
observation. Equations to correct the data for specimen
translation and rotation are developed and 3D reconstructions
of the scala tympani, round window membrane, and cochlear
aqueduct are presented. The error associated with the
reconstruction is estimated to be < 19 microns.
BibTeX:
@article{Voie1995-yn,
  author = {Voie, A H and Spelman, F A},
  title = {Three-dimensional reconstruction of the cochlea from
two-dimensional images of optical sections}, journal = {Comput. Med. Imaging Graph.}, year = {1995}, volume = {19}, number = {5}, pages = {377--384}, url = {http://www.ncbi.nlm.nih.gov/pubmed/8734775} }
Voie, A.H., Spelman, F.A., Sutton, D. and Burns, D.H. Quantitative measurements of the three-dimensional anatomy of
theguinea pig cochlea using optical łdots
1989 Engineering in Medicine and Biology Society  article URL 
Abstract: Page 1. Open Competition I Quantitative Measurements of the
Three-Dimensional Anatomy of the Guinea Pig Cochlea Using Optical
Sectioning
BibTeX:
@article{Voie1989-gf,
  author = {Voie, A H and Spelman, F A and Sutton, D and Burns, D H},
  title = {Quantitative measurements of the three-dimensional anatomy of
theguinea pig cochlea using optical łdots}, journal = {Engineering in Medicine and Biology Society}, year = {1989}, url = {http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=96444} }
Weber, M. and Huisken, J. Omnidirectional microscopy 2012 Nat. Methods
Vol. 9(7), pp. 656-657 
article DOI URL 
BibTeX:
@article{Weber2012-gp,
  author = {Weber, Michael and Huisken, Jan},
  title = {Omnidirectional microscopy},
  journal = {Nat. Methods},
  year = {2012},
  volume = {9},
  number = {7},
  pages = {656--657},
  url = {http://dx.doi.org/10.1038/nmeth.2022},
  doi = {http://dx.doi.org/10.1038/nmeth.2022}
}
Weber, M. and Huisken, J. Light sheet microscopy for real-time developmental biology 2011 Curr. Opin. Genet. Dev.
Vol. 21(5), pp. 566-572 
article DOI URL 
Abstract: Within only a few short years, light sheet microscopy has
contributed substantially to the emerging field of real-time
developmental biology. Low photo-toxicity and high-speed
multiview acquisition have made selective plane illumination
microscopy (SPIM) a popular choice for studies of organ
morphogenesis and function in zebrafish, Drosophila, and other
model organisms. A multitude of different light sheet
microscopes have emerged for the noninvasive imaging of
specimens ranging from single molecules to cells, tissues, and
entire embryos. In particular, developmental biology can
benefit from the ability to watch developmental events occur
in real time in an entire embryo, thereby advancing our
understanding on how cells form tissues and organs. However,
it presents a new challenge to our existing data and image
processing tools. This review gives an overview of where we
stand as light sheet microscopy branches out, explores new
areas, and becomes more specialized.
BibTeX:
@article{Weber2011-ap,
  author = {Weber, Michael and Huisken, Jan},
  title = {Light sheet microscopy for real-time developmental biology},
  journal = {Curr. Opin. Genet. Dev.},
  year = {2011},
  volume = {21},
  number = {5},
  pages = {566--572},
  url = {http://dx.doi.org/10.1016/j.gde.2011.09.009},
  doi = {http://dx.doi.org/10.1016/j.gde.2011.09.009}
}
Weber, M., Mickoleit, M. and Huisken, J. Light sheet microscopy 2014 Methods Cell Biol.
Vol. 123, pp. 193-215 
article DOI URL 
Abstract: This chapter introduces the concept of light sheet microscopy
along with practical advice on how to design and build such an
instrument. Selective plane illumination microscopy is
presented as an alternative to confocal microscopy due to
several superior features such as high-speed full-frame
acquisition, minimal phototoxicity, and multiview sample
rotation. Based on our experience over the last 10 years, we
summarize the key concepts in light sheet microscopy, typical
implementations, and successful applications. In particular,
sample mounting for long time-lapse imaging and the resulting
challenges in data processing are discussed in detail.
BibTeX:
@article{Weber2014-cc,
  author = {Weber, Michael and Mickoleit, Michaela and Huisken, Jan},
  title = {Light sheet microscopy},
  journal = {Methods Cell Biol.},
  year = {2014},
  volume = {123},
  pages = {193--215},
  url = {http://dx.doi.org/10.1016/B978-0-12-420138-5.00011-2},
  doi = {http://dx.doi.org/10.1016/B978-0-12-420138-5.00011-2}
}
Weber, M., Mickoleit, M. and Huisken, J. Multilayer mounting for long-term light sheet microscopy of
zebrafish
2014 J. Vis. Exp.(84), pp. e51119  article DOI URL 
Abstract: Light sheet microscopy is the ideal imaging technique to study
zebrafish embryonic development. Due to minimal photo-toxicity
and bleaching, it is particularly suited for long-term
time-lapse imaging over many hours up to several days.
However, an appropriate sample mounting strategy is needed
that offers both confinement and normal development of the
sample. Multilayer mounting, a new embedding technique using
low-concentration agarose in optically clear tubes, now
overcomes this limitation and unleashes the full potential of
light sheet microscopy for real-time developmental biology.
BibTeX:
@article{Weber2014-yg,
  author = {Weber, Michael and Mickoleit, Michaela and Huisken, Jan},
  title = {Multilayer mounting for long-term light sheet microscopy of
zebrafish}, journal = {J. Vis. Exp.}, year = {2014}, number = {84}, pages = {e51119}, url = {http://dx.doi.org/10.3791/51119}, doi = {http://dx.doi.org/10.3791/51119} }
Weber, T.G., Osl, F., Renner, A., Pöschinger, T., Galbán, S., Rehemtulla, A. and Scheuer, W. Apoptosis imaging for monitoring DR5 antibody accumulation
and pharmacodynamics in brain tumors noninvasively
2014 Cancer Res.
Vol. 74(7), pp. 1913-1923 
article DOI URL 
Abstract: High-grade gliomas often possess an impaired blood-brain
barrier (BBB), which allows delivery of large molecules to
brain tumors. However, achieving optimal drug concentrations
in brain tumors remains a significant hurdle for treating
patients successfully. Thus, detailed investigations of drug
activities in gliomas are needed. To investigate BBB
penetration, pharmacodynamics, and tumor retention kinetics of
an agonistic DR5 antibody in a brain tumor xenograft model, we
utilized a noninvasive imaging method for longitudinal
monitoring of apoptosis induction. Brain tumors were induced
by intracranial (i.c.) implantation of a luciferase-expressing
tumor cell line as a reporter. To quantify accumulation of
anti-DR5 in brain tumors, we generated a dosage-response curve
for apoptosis induction after i.c. delivery of
fluorescence-labeled anti-DR5 at different dosages. Assuming
100% drug delivery after i.c. application, the amount of
accumulated antibody after i.v. application was calculated
relative to its apoptosis induction. We found that up to
0.20% to 0.97% of antibody delivered i.v. reached the brain
tumor, but that apoptosis induction declined quickly within 24
hours. These results were confirmed by three-dimensional
fluorescence microscopy of antibody accumulation in explanted
brains. Nonetheless, significant antitumor efficacy was
documented after anti-DR5 delivery. We further demonstrated
that antibody penetration was facilitated by an impaired BBB
in brain tumors. These imaging methods enable the
quantification of antibody accumulation and pharmacodynamics
in brain tumors, offering a holistic approach for assessment
of central nervous system-targeting drugs.
BibTeX:
@article{Weber2014-uh,
  author = {Weber, Thomas G and Osl, Franz and Renner, Anja and
Pöschinger, Thomas and Galbán, Stefanie and
Rehemtulla, Alnawaz and Scheuer, Werner}, title = {Apoptosis imaging for monitoring DR5 antibody accumulation
and pharmacodynamics in brain tumors noninvasively}, journal = {Cancer Res.}, year = {2014}, volume = {74}, number = {7}, pages = {1913--1923}, url = {http://dx.doi.org/10.1158/0008-5472.CAN-13-3001}, doi = {http://dx.doi.org/10.1158/0008-5472.CAN-13-3001} }
Wein, W., Blume, M., Leischner, U., Dodt, H.-U. and Navab, N. Quality-based registration and reconstruction of optical
tomography volumes
2007 Med. Image Comput. Comput. Assist. Interv.
Vol. 10(Pt 2), pp. 718-725 
article URL 
Abstract: Ultramicroscopy, a novel optical tomographic imaging modality
related to fluorescence microscopy, allows to acquire
cross-sectional slices of small specially prepared biological
samples with astounding quality and resolution. However,
scattering of the fluorescence light causes the quality to
decrease proportional to the depth of the currently imaged
plane. Scattering and beam thickness of the excitation laser
light cause additional image degradation. We perform a
physical simulation of the light scattering in order to define
a quantitative function of image quality with respect to
depth. This allows us to establish 3D-volumes of quality
information in addition to the image data. Volumes are
acquired at different orientations of the sample, hence
providing complementary regions of high quality. We propose an
algorithm for rigid 3D-3D registration of these volumes
incorporating voxel quality information, based on maximizing
an adapted linear correlation term. The quality ratio of the
images is then used, along with the registration result, to
create improved volumes of the imaged object. The methods are
applied on acquisitions of a mouse brain and mouse embryo to
create outstanding three-dimensional reconstructions.
BibTeX:
@article{Wein2007-ce,
  author = {Wein, Wolfgang and Blume, Moritz and Leischner, Ulrich and
Dodt, Hans-Ulrich and Navab, Nassir}, title = {Quality-based registration and reconstruction of optical
tomography volumes}, journal = {Med. Image Comput. Comput. Assist. Interv.}, year = {2007}, volume = {10}, number = {Pt 2}, pages = {718--725}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18044632} }
Wohland, T., Shi, X., Sankaran, J. and Stelzer, E.H.K. Single plane illumination fluorescence correlation
spectroscopy (SPIM-FCS) probes inhomogeneous
three-dimensional environments
2010 Opt. Express
Vol. 18(10), pp. 10627-10641 
article DOI URL 
Abstract: The life sciences require new highly sensitive imaging tools,
which allow the quantitative measurement of molecular
parameters within a physiological three-dimensional (3D)
environment. Therefore, we combined single plane illumination
microscopy (SPIM) with camera based fluorescence correlation
spectroscopy (FCS). SPIM-FCS provides contiguous particle
number and diffusion coefficient images with a high spatial
resolution in homo- and heterogeneous 3D specimens and live
zebrafish embryos. Our SPIM-FCS recorded up to 4096 spectra
within 56 seconds at a laser power of 60 microW without
damaging the embryo. This new FCS modality provides more
measurements per time and more, less photo-toxic measurements
per sample than confocal based methods. In essence, SPIM-FCS
offers new opportunities to observe biomolecular interactions
quantitatively and functions in a highly multiplexed manner
within a physiologically relevant 3D environment.
BibTeX:
@article{Wohland2010-ck,
  author = {Wohland, Thorsten and Shi, Xianke and Sankaran, Jagadish and
Stelzer, Ernst H K}, title = {Single plane illumination fluorescence correlation
spectroscopy (SPIM-FCS) probes inhomogeneous
three-dimensional environments}, journal = {Opt. Express}, year = {2010}, volume = {18}, number = {10}, pages = {10627--10641}, url = {http://dx.doi.org/10.1364/OE.18.010627}, doi = {http://dx.doi.org/10.1364/OE.18.010627} }
Wolf, S., Supatto, W., Debrégeas, G., Mahou, P., Kruglik, S.G., Sintes, J.-M., Beaurepaire, E. and Candelier, R. Whole-brain functional imaging with two-photon light-sheet
microscopy
2015 Nat. Methods
Vol. 12(5), pp. 379-380 
article DOI URL 
BibTeX:
@article{Wolf2015-tp,
  author = {Wolf, Sébastien and Supatto, Willy and Debrégeas,
Georges and Mahou, Pierre and Kruglik, Sergei G and Sintes,
Jean-Marc and Beaurepaire, Emmanuel and Candelier,
Raphaël}, title = {Whole-brain functional imaging with two-photon light-sheet
microscopy}, journal = {Nat. Methods}, year = {2015}, volume = {12}, number = {5}, pages = {379--380}, url = {http://dx.doi.org/10.1038/nmeth.3371}, doi = {http://dx.doi.org/10.1038/nmeth.3371} }
Wu, J., Li, J. and Chan, R.K.Y. A light sheet based high throughput 3D-imaging flow cytometer
for phytoplankton analysis
2013 Opt. Express
Vol. 21(12), pp. 14474-14480 
article DOI URL 
Abstract: This paper reports a light sheet fluorescence imaging flow
cytometer for 3D sectioning of phytoplankton. The instrument
developed has the inherent advantages of high cell counting
throughput and high spatial resolution information derived
from flow cytometry and light sheet microscopy. The throughput
of the instrument is quantified by the sample volume flow rate
of 0.5 $l/min with a spatial resolution as achieved by
light sheet microscopy. Preliminary results from 3D morphology
of the internal chlorophyll-a structure of two dinoflagellates
species show promising application potentials of the method
for phytoplankton taxonomy of selected species and species
groups.
BibTeX:
@article{Wu2013-hb,
  author = {Wu, Jianglai and Li, Jianping and Chan, Robert K Y},
  title = {A light sheet based high throughput 3D-imaging flow cytometer
for phytoplankton analysis}, journal = {Opt. Express}, year = {2013}, volume = {21}, number = {12}, pages = {14474--14480}, url = {http://dx.doi.org/10.1364/OE.21.014474}, doi = {http://dx.doi.org/10.1364/OE.21.014474} }
Wu, T.-F., Yen, T.M., Han, Y., Chiu, Y.-J., Lin, E.Y.-S. and Lo, Y.-H. A light-sheet microscope compatible with mobile devices for
label-free intracellular imaging and biosensing
2014 Lab Chip
Vol. 14(17), pp. 3341-3348 
article DOI URL 
Abstract: The inner structure, especially the nuclear structure, of cells
carries valuable information about disease and health conditions
of a person. Here we demonstrate a label-free technique to
enable direct observations and measurements of the size, shape
and morphology of the cell nucleus. With a microfabricated lens
and a commercial CMOS imager, we form a scanning light-sheet
microscope to produce a dark-field optical scattering image of
the cell nucleus that overlays with the bright-field image
produced in a separate regime of the same CMOS sensor. We have
used the device to detect nuclear features that characterize the
life cycle of cells and have used the nucleus volume as a new
parameter for cell classification. The device can be developed
into a portable, low-cost, point-of-care device leveraging the
capabilities of the CMOS imagers to be pervasive in mobile
electronics.
BibTeX:
@article{Wu2014-fp,
  author = {Wu, Tsung-Feng and Yen, Tony Minghung and Han, Yuanyuan and
Chiu, Yu-Jui and Lin, Eason Y-S and Lo, Yu-Hwa}, title = {A light-sheet microscope compatible with mobile devices for
label-free intracellular imaging and biosensing}, journal = {Lab Chip}, publisher = {The Royal Society of Chemistry}, year = {2014}, volume = {14}, number = {17}, pages = {3341--3348}, url = {http://pubs.rsc.org/en/Content/ArticleLanding/2014/LC/C4LC00257A}, doi = {http://dx.doi.org/10.1039/C4LC00257A} }
Wu, Y., Ghitani, A., Christensen, R., Santella, A., Du, Z., Rondeau, G., Bao, Z., Colón-Ramos, D. and Shroff, H. Inverted selective plane illumination microscopy (iSPIM)
enables coupled cell identity lineaging and neurodevelopmental
imaging in Caenorhabditis elegans
2011 Proc. Natl. Acad. Sci. U. S. A.
Vol. 108(43), pp. 17708-17713 
article DOI URL 
Abstract: The Caenorhabditis elegans embryo is a powerful model for
studying neural development, but conventional imaging methods
are either too slow or phototoxic to take full advantage of
this system. To solve these problems, we developed an inverted
selective plane illumination microscopy (iSPIM) module for
noninvasive high-speed volumetric imaging of living samples.
iSPIM is designed as a straightforward add-on to an inverted
microscope, permitting conventional mounting of specimens and
facilitating SPIM use by development and neurobiology
laboratories. iSPIM offers a volumetric imaging rate
30 faster than currently used technologies, such
as spinning-disk confocal microscopy, at comparable
signal-to-noise ratio. This increased imaging speed allows us
to continuously monitor the development of C, elegans embryos,
scanning volumes every 2 s for the 14-h period of
embryogenesis with no detectable phototoxicity. Collecting
25,000 volumes over the entirety of embryogenesis enabled in
toto visualization of positions and identities of cell nuclei.
By merging two-color iSPIM with automated lineaging techniques
we realized two goals: (i) identification of neurons
expressing the transcription factor CEH-10/Chx10 and (ii)
visualization of their neurodevelopmental dynamics. We found
that canal-associated neurons use somal translocation and
amoeboid movement as they migrate to their final position in
the embryo. We also visualized axon guidance and growth cone
dynamics as neurons circumnavigate the nerve ring and reach
their targets in the embryo. The high-speed volumetric imaging
rate of iSPIM effectively eliminates motion blur from embryo
movement inside the egg case, allowing characterization of
dynamic neurodevelopmental events that were previously
inaccessible.
BibTeX:
@article{Wu2011-md,
  author = {Wu, Yicong and Ghitani, Alireza and Christensen, Ryan and
Santella, Anthony and Du, Zhuo and Rondeau, Gary and Bao,
Zhirong and Colón-Ramos, Daniel and Shroff, Hari}, title = {Inverted selective plane illumination microscopy (iSPIM)
enables coupled cell identity lineaging and neurodevelopmental
imaging in Caenorhabditis elegans}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, year = {2011}, volume = {108}, number = {43}, pages = {17708--17713}, url = {http://dx.doi.org/10.1073/pnas.1108494108}, doi = {http://dx.doi.org/10.1073/pnas.1108494108} }
Wu, Y., Wawrzusin, P., Senseney, J., Fischer, R.S., Christensen, R., Santella, A., York, A.G., Winter, P.W., Waterman, C.M., Bao, Z., Colón-Ramos, D.A., McAuliffe, M. and Shroff, H. Spatially isotropic four-dimensional imaging with dual-view
plane illumination microscopy
2013 Nat. Biotechnol.
Vol. 31(11), pp. 1032-1038 
article DOI URL 
Abstract: Optimal four-dimensional imaging requires high spatial
resolution in all dimensions, high speed and minimal
photobleaching and damage. We developed a dual-view, plane
illumination microscope with improved spatiotemporal
resolution by switching illumination and detection between two
perpendicular objectives in an alternating duty cycle.
Computationally fusing the resulting volumetric views provides
an isotropic resolution of 330 nm. As the sample is stationary
and only two views are required, we achieve an imaging speed
of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike
spinning-disk confocal or Bessel beam methods, which
illuminate the sample outside the focal plane, we maintain
high spatiotemporal resolution over hundreds of volumes with
negligible photobleaching. To illustrate the ability of our
method to study biological systems that require high-speed
volumetric visualization and/or low photobleaching, we
describe microtubule tracking in live cells, nuclear imaging
over 14 h during nematode embryogenesis and imaging of neural
wiring during Caenorhabditis elegans brain development over 5
h.
BibTeX:
@article{Wu2013-pv,
  author = {Wu, Yicong and Wawrzusin, Peter and Senseney, Justin and
Fischer, Robert S and Christensen, Ryan and Santella, Anthony
and York, Andrew G and Winter, Peter W and Waterman, Clare M
and Bao, Zhirong and Colón-Ramos, Daniel A and McAuliffe,
Matthew and Shroff, Hari}, title = {Spatially isotropic four-dimensional imaging with dual-view
plane illumination microscopy}, journal = {Nat. Biotechnol.}, year = {2013}, volume = {31}, number = {11}, pages = {1032--1038}, url = {http://dx.doi.org/10.1038/nbt.2713}, doi = {http://dx.doi.org/10.1038/nbt.2713} }
Xu, Z. and Holy, T.E. Development of low-coherence light sheet illumination microscope
for fluorescence-free bioimaging
2011 SPIE Optical Engineering + Applications, pp. 812908-812908-10  inproceedings DOI URL 
Abstract: abstract Light Sheet Illumination Microscopy (LSIM) is an
imaging modality featuring the novel arrangement with the
illumination axis perpendicular to the detection axis. In this
technology a well defined light sheet is generated and aligned
precisely to the focal plane ...
BibTeX:
@inproceedings{Xu2011-xq,
  author = {Xu, Zhiguang and Holy, Timothy E},
  title = {Development of low-coherence light sheet illumination microscope
for fluorescence-free bioimaging}, booktitle = {SPIE Optical Engineering + Applications}, publisher = {International Society for Optics and Photonics}, year = {2011}, pages = {812908--812908--10}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1266985}, doi = {http://dx.doi.org/10.1117/12.893569} }
Yang, Z., Mei, L., Xia, F., Luo, Q., Fu, L. and Gong, H. Dual-slit confocal light sheet microscopy for in vivo
whole-brain imaging of zebrafish
2015 Biomed. Opt. Express
Vol. 6(5), pp. 1797-1811 
article DOI URL 
Abstract: In vivo functional imaging at single-neuron resolution is an
important approach to visualize biological processes in
neuroscience. Light sheet microscopy (LSM) is a cutting edge
in vivo imaging technique that provides micron-scale spatial
resolution at high frame rate. Due to the scattering and
absorption of tissue, however, conventional LSM is inadequate
to resolve cells because of the attenuated signal to noise
ratio (SNR). Using dual-beam illumination and confocal
dual-slit detection, here a dual-slit confocal LSM is
demonstrated to obtain the SNR enhanced images with frame rate
twice as high as line confocal LSM method. Through theoretical
calculations and experiments, the correlation between the
slit's width and SNR was determined to optimize the image
quality. In vivo whole brain structural imaging stacks and the
functional imaging sequences of single slice were obtained for
analysis of calcium activities at single-cell resolution. A
two-fold increase in imaging speed of conventional confocal
LSM makes it possible to capture the sequence of the neurons'
activities and help reveal the potential functional
connections in the whole zebrafish's brain.
BibTeX:
@article{Yang2015-gk,
  author = {Yang, Zhe and Mei, Li and Xia, Fei and Luo, Qingming and Fu,
Ling and Gong, Hui}, title = {Dual-slit confocal light sheet microscopy for in vivo
whole-brain imaging of zebrafish}, journal = {Biomed. Opt. Express}, year = {2015}, volume = {6}, number = {5}, pages = {1797--1811}, url = {http://dx.doi.org/10.1364/BOE.6.001797}, doi = {http://dx.doi.org/10.1364/BOE.6.001797} }
Yang, Z., Piksarv, P., Ferrier, D.E.K., Gunn-Moore, F.J. and Dholakia, K. Macro-optical trapping for sample confinement in light sheet
microscopy
2015 Biomed. Opt. Express
Vol. 6(8), pp. 2778-2785 
article DOI URL 
Abstract: Light sheet microscopy is a powerful approach to construct
three-dimensional images of large specimens with minimal
photo-damage and photo-bleaching. To date, the specimens are
usually mounted in agents such as agarose, potentially
restricting the development of live samples, and also highly
mobile specimens need to be anaesthetized before imaging. To
overcome these problems, here we demonstrate an integrated
light sheet microscope which solely uses optical forces to
trap and hold the sample using a counter-propagating laser
beam geometry. Specifically, tobacco plant cells and living
Spirobranchus lamarcki larvae were successfully trapped and
sectional images acquired. This novel approach has the
potential to significantly expand the range of applications
for light sheet imaging.
BibTeX:
@article{Yang2015-lb,
  author = {Yang, Zhengyi and Piksarv, Peeter and Ferrier, David E K and
Gunn-Moore, Frank J and Dholakia, Kishan}, title = {Macro-optical trapping for sample confinement in light sheet
microscopy}, journal = {Biomed. Opt. Express}, year = {2015}, volume = {6}, number = {8}, pages = {2778--2785}, url = {http://dx.doi.org/10.1364/BOE.6.002778}, doi = {http://dx.doi.org/10.1364/BOE.6.002778} }
Yang, Z., Piksarv, P., Ferrier, D.E.K., Gunn-Moore, F.J. and Dholakia, K. Integrated 3D macro-trapping and light-sheet imaging system 2015 SPIE Nanoscience + Engineering, pp. 95480T-95480T-6  inproceedings DOI URL 
BibTeX:
@inproceedings{Yang2015-qt,
  author = {Yang, Zhengyi and Piksarv, Peeter and Ferrier, David E K and
Gunn-Moore, Frank J and Dholakia, Kishan}, title = {Integrated 3D macro-trapping and light-sheet imaging system}, booktitle = {SPIE Nanoscience + Engineering}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {95480T--95480T--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2432035}, doi = {http://dx.doi.org/10.1117/12.2186399} }
Yang, Z., Prokopas, M., Nylk, J., Coll-Lladó, C., Gunn-Moore, F.J., Ferrier, D.E.K., Vettenburg, T. and Dholakia, K. A compact Airy beam light sheet microscope with a tilted
cylindrical lens
2014 Biomed. Opt. Express
Vol. 5(10), pp. 3434-3442 
article DOI URL 
Abstract: Light-sheet imaging is rapidly gaining importance for imaging
intact biological specimens. Many of the latest innovations
rely on the propagation-invariant Bessel or Airy beams to form
an extended light sheet to provide high resolution across a
large field of view. Shaping light to realize
propagation-invariant beams often relies on complex
programming of spatial light modulators or specialized, custom
made, optical elements. Here we present a straightforward and
low-cost modification to the traditional light-sheet setup,
based on the open-access light-sheet microscope OpenSPIM, to
achieve Airy light-sheet illumination. This brings wide field
single-photon light-sheet imaging to a broader range of
endusers. Fluorescent microspheres embedded in agarose and a
zebrafish larva were imaged to demonstrate how such a
microscope can have a minimal footprint and cost without
compromising on imaging quality.
BibTeX:
@article{Yang2014-cn,
  author = {Yang, Zhengyi and Prokopas, Martynas and Nylk, Jonathan and
Coll-Lladó, Clara and Gunn-Moore, Frank J and Ferrier,
David E K and Vettenburg, Tom and Dholakia, Kishan}, title = {A compact Airy beam light sheet microscope with a tilted
cylindrical lens}, journal = {Biomed. Opt. Express}, publisher = {Optical Society of America}, year = {2014}, volume = {5}, number = {10}, pages = {3434--3442}, url = {http://dx.doi.org/10.1364/BOE.5.003434}, doi = {http://dx.doi.org/10.1364/BOE.5.003434} }
Young, L.K., Jarrin, M., Saunter, C.D., Quinlan, R. and Girkin, J.M. Using SPIM to track the development of the focal power of the
zebrafish lens
2015 SPIE BiOS, pp. 933408-933408-7  inproceedings DOI URL 
BibTeX:
@inproceedings{Young2015-dt,
  author = {Young, Laura K and Jarrin, Miguel and Saunter, Christopher D and
Quinlan, Roy and Girkin, John M}, title = {Using SPIM to track the development of the focal power of the
zebrafish lens}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2015}, pages = {933408--933408--7}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=2203123}, doi = {http://dx.doi.org/10.1117/12.2079887} }
Zapata-Rodrguez, C.J., Martnez-Corral, M. and Mu noz-Escrivá, L. Axial Superresolution by Oblique Two-Dimensional
Nondiffracting cos Beam Illumination
1998 J. Comput. Assist. Microsc.
Vol. 10(3), pp. 97-101 
article DOI URL 
Abstract: Nondiffracting cos beams may be used in the object space of an
optical microscope for causing a nonuniform illumination. This
irradiance distribution consists in a set of equidistant plane
maxima, and therefore the light radiated by the sample decays in
its neighborhood. We propose to observe over an object plane
coinciding with one of these illumination peaks, which results
in a superresolving axial effect. For that purpose, illumination
and detection should be oblique processes, and a
computer-assisted z-scanning process is needed in order to
access the axial structure of a thick object.
BibTeX:
@article{Zapata-Rodriguez1998-yr,
  author = {Zapata-Rodrguez, Carlos J and Martnez-Corral, Manuel
and Muñoz-Escrivá, Laura}, title = {Axial Superresolution by Oblique Two-Dimensional
Nondiffracting cos Beam Illumination}, journal = {J. Comput. Assist. Microsc.}, publisher = {Kluwer Academic Publishers-Plenum Publishers}, year = {1998}, volume = {10}, number = {3}, pages = {97--101}, url = {http://link.springer.com/article/10.1023/A%3A1023447018678}, doi = {http://dx.doi.org/10.1023/A:1023447018678} }
Zhang, P., Goodwin, P.M. and Werner, J.H. Fast, 3D imaging via confocal line scanning of a Bessel beam
using a single galvo mirror
2014 SPIE BiOS, pp. 89471K-89471K-8  inproceedings DOI URL 
BibTeX:
@inproceedings{Zhang2014-lg,
  author = {Zhang, Pengfei and Goodwin, Peter M and Werner, James H},
  title = {Fast, 3D imaging via confocal line scanning of a Bessel beam
using a single galvo mirror}, booktitle = {SPIE BiOS}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {89471K--89471K--8}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1840817}, doi = {http://dx.doi.org/10.1117/12.2036848} }
Zhang, P., Goodwin, P.M. and Werner, J.H. Fast, super resolution imaging via Bessel-beam stimulated
emission depletion microscopy
2014 Opt. Express
Vol. 22(10), pp. 12398-12409 
article DOI URL 
Abstract: A substantial advantage of stimulated emission depletion (STED)
microscopy over other super-resolution methods is that images
can be acquired in real-time without any post-processing.
However imaging speed and photodamage are two major concerns for
STED imaging of whole cells. Here we propose a new microscopy
method we have termed Bessel-Beam STED (or BB-STED) that
overcomes both of these limitations of conventional STED
microscopy. In the proposed method, rather than exciting a
single STED spot in the sample, an entire line of the sample is
illuminated. This line-scanning technique dramatically increases
the speed of STED. In addition, plane-illumination by scanning
of the line across the focal plane of a detection objective
limits the light to a thin layer of the sample and thus
significantly reduces photobleaching and photodamage above and
below the focal plane compared to epi-illumination. Using the
organic dye Atto647N as an example, we calculated the STED power
required to break the diffraction limit. The results presented
here will be used to guide future experimental designs.
BibTeX:
@article{Zhang2014-xy,
  author = {Zhang, P and Goodwin, P M and Werner, J H},
  title = {Fast, super resolution imaging via Bessel-beam stimulated
emission depletion microscopy}, journal = {Opt. Express}, publisher = {Optical Society of America}, year = {2014}, volume = {22}, number = {10}, pages = {12398--12409}, url = {http://dx.doi.org/10.1364/OE.22.012398}, doi = {http://dx.doi.org/10.1364/OE.22.012398} }
Zhang, P., Phipps, M.E., Goodwin, P.M. and Werner, J.H. Confocal line scanning of a Bessel beam for fast 3D imaging 2014 Opt. Lett.
Vol. 39(12), pp. 3682-3685 
article DOI URL 
Abstract: We have developed a light-sheet illumination microscope that can
perform fast 3D imaging of transparent biological samples with
inexpensive visible lasers and a single galvo mirror (GM). The
light-sheet is created by raster scanning a Bessel beam with a
GM, with this same GM also being used to rescan the fluorescence
across a chip of a camera to construct an image in real time. A
slit is used to reject out-of-focus fluorescence such that the
image formed in real time has minimal contribution from the
sidelobes of the Bessel beam. Compared with two-photon Bessel
beam excitation or other confocal line-scanning approaches, our
method is of lower cost, is simpler, and does not require
calibration and synchronization of multiple GMs. We demonstrated
the optical sectioning and out-of-focus background rejection
capabilities of this microscope by imaging fluorescently labeled
actin filaments in fixed 3T3 cells.
BibTeX:
@article{Zhang2014-of,
  author = {Zhang, P and Phipps, M E and Goodwin, P M and Werner, J H},
  title = {Confocal line scanning of a Bessel beam for fast 3D imaging},
  journal = {Opt. Lett.},
  year = {2014},
  volume = {39},
  number = {12},
  pages = {3682--3685},
  url = {http://dx.doi.org/10.1364/OL.39.003682},
  doi = {http://dx.doi.org/10.1364/OL.39.003682}
}
Zhao, M., Zhang, H., Li, Y., Ashok, A., Liang, R., Zhou, W. and Peng, L. Cellular imaging of deep organ using two-photon Bessel
light-sheet nonlinear structured illumination microscopy
2014 Biomed. Opt. Express
Vol. 5(5), pp. 1296-1308 
article DOI URL 
Abstract: In vivo fluorescent cellular imaging of deep internal organs
is highly challenging, because the excitation needs to
penetrate through strong scattering tissue and the emission
signal is degraded significantly by photon diffusion induced
by tissue-scattering. We report that by combining two-photon
Bessel light-sheet microscopy with nonlinear structured
illumination microscopy (SIM), live samples up to 600 microns
wide can be imaged by light-sheet microscopy with 500 microns
penetration depth, and diffused background in deep tissue
light-sheet imaging can be reduced to obtain clear images at
cellular resolution in depth beyond 200 microns. We
demonstrate in vivo two-color imaging of pronephric glomeruli
and vasculature of zebrafish kidney, whose cellular structures
located at the center of the fish body are revealed in high
clarity by two-color two-photon Bessel light-sheet SIM.
BibTeX:
@article{Zhao2014-mw,
  author = {Zhao, Ming and Zhang, Han and Li, Yu and Ashok, Amit and
Liang, Rongguang and Zhou, Weibin and Peng, Leilei}, title = {Cellular imaging of deep organ using two-photon Bessel
light-sheet nonlinear structured illumination microscopy}, journal = {Biomed. Opt. Express}, publisher = {Optical Society of America}, year = {2014}, volume = {5}, number = {5}, pages = {1296--1308}, url = {http://dx.doi.org/10.1364/BOE.5.001296}, doi = {http://dx.doi.org/10.1364/BOE.5.001296} }
Zhao, Z.W., Roy, R., Gebhardt, J.C.M., Suter, D.M., Chapman, A.R. and Xie, X.S. Spatial organization of RNA polymerase II inside a
mammalian cell nucleus revealed by reflected light-sheet
superresolution microscopy
2014 Proc. Natl. Acad. Sci. U. S. A.
Vol. 111(2), pp. 681-686 
article DOI URL 
Abstract: Superresolution microscopy based on single-molecule centroid
determination has been widely applied to cellular imaging in
recent years. However, quantitative imaging of the mammalian
nucleus has been challenging due to the lack of 3D optical
sectioning methods for normal-sized cells, as well as the
inability to accurately count the absolute copy numbers of
biomolecules in highly dense structures. Here we report a
reflected light-sheet superresolution microscopy method
capable of imaging inside the mammalian nucleus with superior
signal-to-background ratio as well as molecular counting with
single-copy accuracy. Using reflected light-sheet
superresolution microscopy, we probed the spatial organization
of transcription by RNA polymerase II (RNAP II) molecules and
quantified their global extent of clustering inside the
mammalian nucleus. Spatiotemporal clustering analysis that
leverages on the blinking photophysics of specific organic
dyes showed that the majority (>70 of the transcription
foci originate from single RNAP II molecules, and no
significant clustering between RNAP II molecules was detected
within the length scale of the reported diameter of
``transcription factories.'' Colocalization measurements of
RNAP II molecules equally labeled by two spectrally distinct
dyes confirmed the primarily unclustered distribution, arguing
against a prevalent existence of transcription factories in
the mammalian nucleus as previously proposed. The methods
developed in our study pave the way for quantitative mapping
and stoichiometric characterization of key biomolecular
species deep inside mammalian cells.
BibTeX:
@article{Zhao2014-nt,
  author = {Zhao, Ziqing W and Roy, Rahul and Gebhardt, J Christof M and
Suter, David M and Chapman, Alec R and Xie, X Sunney}, title = {Spatial organization of RNA polymerase II inside a
mammalian cell nucleus revealed by reflected light-sheet
superresolution microscopy}, journal = {Proc. Natl. Acad. Sci. U. S. A.}, year = {2014}, volume = {111}, number = {2}, pages = {681--686}, url = {http://dx.doi.org/10.1073/pnas.1318496111}, doi = {http://dx.doi.org/10.1073/pnas.1318496111} }
Zong, W., Zhao, J., Chen, X., Lin, Y., Ren, H., Zhang, Y., Fan, M., Zhou, Z., Cheng, H., Sun, Y. and Chen, L. Large-field high-resolution two-photon digital scanned
light-sheet microscopy
2015 Cell Res.
Vol. 25(2), pp. 254-257 
article DOI URL 
Abstract: Cell death and differentiation is a monthly research journal
focused on the exciting field of programmed cell death and
apoptosis. It provides a single accessible source of
information for both scientists and clinicians, keeping them
up-to-date with advances in the field. It encompasses
programmed cell death, cell death induced by toxic agents,
differentiation and the interrelation of these with cell
proliferation.
BibTeX:
@article{Zong2015-xl,
  author = {Zong, Weijian and Zhao, Jia and Chen, Xuanyang and Lin, Yuan
and Ren, Huixia and Zhang, Yunfeng and Fan, Ming and Zhou,
Zhuan and Cheng, Heping and Sun, Yujie and Chen, Liangyi}, title = {Large-field high-resolution two-photon digital scanned
light-sheet microscopy}, journal = {Cell Res.}, publisher = {Nature Publishing Group}, year = {2015}, volume = {25}, number = {2}, pages = {254--257}, url = {http://dx.doi.org/10.1038/cr.2014.124}, doi = {http://dx.doi.org/10.1038/cr.2014.124} }
Zong, W., Zhao, J., Chen, X., Lin, Y., Ren, H., Zhang, Y., Fan, M., Zhou, Z., Cheng, H., Sun, Y. and Chen, L. Two-photon three-axis digital scanned light-sheet microscopy
(2P3A-DSLM)
2014 SPIE Optical Engineering + Applications, pp. 91980W-91980W-6  inproceedings DOI URL 
Abstract: abstract In this presentation we report a new 3D scanned DSLM.
The system combined 1) two-photon excitation, 2) scanning along
the illumination axis (x-axis) using tunable acoustic gradient
lens (TAG) to stretch the Rayleigh range [5], 3) scanning
vertically to the ...
BibTeX:
@inproceedings{Zong2014-is,
  author = {Zong, Weijian and Zhao, Jia and Chen, Xuanyang and Lin, Yuan and
Ren, Huixia and Zhang, Yunfeng and Fan, Ming and Zhou, Zhuan and
Cheng, Heping and Sun, Yujie and Chen, Liangyi}, title = {Two-photon three-axis digital scanned light-sheet microscopy
(2P3A-DSLM)}, booktitle = {SPIE Optical Engineering + Applications}, publisher = {International Society for Optics and Photonics}, year = {2014}, pages = {91980W--91980W--6}, url = {http://proceedings.spiedigitallibrary.org/proceeding.aspx?articleid=1916125}, doi = {http://dx.doi.org/10.1117/12.2062690} }
Zsigmondy, R. and Bachmann, W. Handhabung des Immersionsultramikroskops 1914 Kolloid-Zeitschrift
Vol. 14(6), pp. 281-295 
article DOI URL 
BibTeX:
@article{Zsigmondy1914-jc,
  author = {Zsigmondy, R and Bachmann, W},
  title = {Handhabung des Immersionsultramikroskops},
  journal = {Kolloid-Zeitschrift},
  publisher = {Springer-Verlag},
  year = {1914},
  volume = {14},
  number = {6},
  pages = {281--295},
  url = {http://link.springer.com/article/10.1007/BF01423340},
  doi = {http://dx.doi.org/10.1007/BF01423340}
}
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