Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high 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 light sheet fluorescence microscopy scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1,000 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, light sheet fluorescence microscopy 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
Basic setup
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. The fluorescence light emitted from the lightsheet is then collected perpendicularly with a standard microscope objective and projected onto an imaging sensor (usually a CCD, electron-multiplying CCD or CMOS camera). In order to let enough space for the excitation optics/lightsheet an observation objective with high working distance is used. In most light sheet fluorescence microscopes the detection objective and sometimes also the excitation objective are fully immersed in the sample buffer, so usually the sample and excitation/detection optics are embedded into a buffer-filled sample chamber, which can also be used to control the environmental conditions (temperature, carbon dioxide level ...) during the measurement. The sample mounting in light sheet fluorescence microscopy is described below in more detail. As both the excitation lightsheet and the focal plane of the detection optics have to coincide to form an image, focusing different parts of the sample can not be done by translating the detection objective, but usually the whole sample is translated and rotated instead.
Extensions of the basic idea In recent years, several extensions to this scheme have been developed:
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