S09-02 Light sheet based fluorescence microscopes (LSFM, SPIM, DSLM) reduce phototoxic effects by several orders of magnitude

Most biochemical compounds absorb light and suffer phototoxic degradation, which induces malfunction or death of a specimen. The situation is particularly dramatic in conventional and confocal fluorescence microscopy. Even though only a single plane is observed, the entire specimen is illuminated. Recording stacks of images along the optical z-axis thus illuminates the entire specimen once for each plane. Hence cells are illuminated 10–20 and fish embryos even 100–300 times more often than they are observed. Surprisingly, this can be avoided by a simple change of the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and which overlap with the focal plane of a wide-field fluorescence microscope. Thus, in contrast to an epi-fluorescence arrangement an azimuth arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage outside a small volume around the focal plane are intrinsic properties. Light sheet-based fluorescence microscopes (LSFM) take advantage of modern camera technologies with a signal to noise ratio that is at least thirty times better than that of a confocal microscope. LSFM can be combined with essentially every contrast and specimen manipulation tool. In a recent application, they were used to record early zebrafish (Danio rerio) development invivo and in toto from the early 32-cell stage until late neurulation with sub-cellular resolution and 60–90 s sampling periods.