We develop optical single molecule assays based on total-internal reflection fluorescence (TIRF) und fluorescence-interference contrast (FLIC) microscopy. Together with novel fluorescent probes (such as quantum dots) and optimized tracking algorithms, we apply these microscopy techniques to study the interaction of motor proteins with their filamentous substrates with nm-resolution in three dimensions.
In TIRF microscopy only the evanescent layer (thickness about 150 nm) is illuminated by the excitation light. Hence, any background fluorescence from the test sample is suppressed and the imaging of fluorescent objects near the surface becomes possible with high signal-to-noise ratio. Individual fluorophores can then be tracked by mathematical algorithms with a precision in the nm range [1, 2].
FLIC microscopy is based on the self-interference of fluorescent light from objects near a reflecting surface. Thereby, the detected fluoresecence intensity becomes a periodic function of the distance at which the object is located above the surface. Utilizing an in situ calibration method, we determined that kinesin-1 molecules elevate gliding MTs about 20 nm. While this value is significantly shorter than the contour length of the motor molecule (in the range of 70 nm), it is consistent with the segmented structure of the motor [3]. Moreover, we used FLIC microscopy to demonstrate a novel solvent sensitive sensor based on the fluorescence of quantum dots attached to stimuli-responsive polymer brushes [4].
References
[1] J. Helenius, G. Brouhard, Y. Kalaidzidis, S. Diez, J. Howard The depolymerizing kinesin MCAK uses 1D-diffusion to rapidly target the ends of microtubules Nature, vol. 441, 115-119, 2006
[2] C. Leduc, F. Ruhnow, J. Howard, S. Diez Detection of fractional steps in cargo movement by the collective operation of kinesin-1 motors Proc Natl Acad Sci USA, Vol. 104, no. 26, pp. 10847-10852, 2007
[3] J. Kerssemakers, J. Howard, H. Hess, S. Diez The distance that kinesin holds its cargo away from the microtubule surface measured by fluorescence-interference-contrast microscopy Proc Natl Acad Sci USA, Vol. 103, no. 43, pp. 15812-15817, 2006
[4] L. Ionov, A. Synytska, S. Sapra, A. L. Rogach, M. Stamm, S. Diez Fast and spatially resolved enviromental sensing using grafted composite layers of stimuli-responsive polymers and semiconductor nanocrystals Advanced Materials, vol. 18, 1453-1457, 2006
