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 and for microscopy of intact, often chemically cleared, organs, embryos, and 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.

In this type of microscopy, the illumination is done perpendicularly to the direction of observation (see schematic image at the top of the article). The expanded beam of a laser is focused in only one direction by a cylindrical lens, or by a combination of a cylindrical lens and a microscope objective as the latter is available in better optical quality and with higher numerical aperture than the first. This way a thin sheet of light or lightsheet is created in the focal region that can be used to excite fluorescence only in a thin slice (usually a few micrometers thin) of the sample.

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