Single molecule tracking with light sheet microscopy

The work presented here concentrates on light sheet based fluorescence microscopy (LSFM) and its application to single molecule tracking. In LSFM the sample is illuminated perpendicular to the detection axis with a thin light sheet. In this manner a simple optical sectioning microscope is created, because only the focal plane of the detection optics is illuminated and no out-of-focus fluorescence is generated. This results in an enhancement of the signal-to-noise-ratio and combined with the high acquisition speed of a video microscopy a powerful tool is created to study single molecule dynamics on a millisecond timescale, A completely new setup was designed and constructed, that combines light sheet illumination technique with single molecule detection ability. Theoretical calculations and quantitative measurements of the illumination light sheet thickness (2-3 µm thick) and the microscope point spread function were performed. A direct comparison of LSFM and epi-illumination of model samples with intrinsic background fluorescence illustrated the clear contrast improvement of LSFM for thick samples. Single molecule detection is limited by the number of photons emitted by a single fluorophore per observation time. So, the ability to track single molecules is dependent on molecule speed, background, detection sensitivity and frame rate. The imaging speed with the concomitant high signal-to-noise ratio that could be realized within the setup was unprecedented until then. It permitted the observation of single protein trajectories in aqueous solution with a diffusion coefficient greater than 100 µm²/s. The in vivo imaging of single molecules in thick biological samples was demonstrated in living salivary gland cell nuclei of Chironomus tentans larvae. These cell nuclei afford exceptional possibilities for the study of RNA mobility, but provide a microscopic challenge with a diameter of 50-75 µm and up 200 µm deep within the sample. To image the intranuclear mobility of individual messenger RNA particles, they were ...