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light sheet fluorescence microscopy
Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate optical resolution, but good optical sectioning capabilities and high speed. In contrast to epifluorescence microscopy only a thin slice (usually a few hundred nanometers to a few micrometers) of the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser light-sheet is used, i.e. a laser beam which is focused only in one direction (e.g. using a cylindrical lens). A second method uses a circular beam scanned in one direction to create the lightsheet. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because LSFM scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1000 times faster than those offered by point-scanning methods.
This method is used in cell biology〔Philipp J. Keller, Ernst H. K. Stelzer: ''Lichtscheiben-Mikroskopie in der molekularen Zellbiophysik'' In: ''LABORWELT.'' 7. Jahrgang, Nr. 5, 2006, S. 18–21 ((Online-Version )).〕 and for microscopy of whole living creatures, such as embryos.〔U. Krzic, S. Günther, L. Hufnagel, D. von Gegerfelt, H. Karlsson, E. Illy, J. Hel: ''Lichtscheiben-Fluoreszenzmikroskopie (SPIM) und Laser-Anregung in orange zur Abbildung lebender Organismen.'' In: ''BioPhotonik.'' Nr. 1, 2011, S. 42–44 ((Online-Version )).〕 It is now often used for the longtime observation of embryonal development in different model organisms.
Starting in 1994, LSFM was developed as orthogonal plane fluorescence optical sectioning microscopy or tomography (OPFOS) mainly for large samples and later as the selective/single plane illumination microscopy (SPIM) also with sub-cellular resolution. This introduced an illumination scheme into fluorescence microscopy, which has already been used successfully for dark field microscopy under the name ultramicroscopy.
== Setup of LSFM ==
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