Legend: CAM=camera, TL=tube lens, F=filter, DO=detection objective, S=sample, SC=sample chamber, PO=projection objective, CL=cylindrical lens, SM=scanning mirror Setup Basic setup Illustration of different light sheet fluorescence microscope implementations. This introduced an illumination scheme into fluorescence microscopy, which has already been used successfully for dark field microscopy under the name ultramicroscopy. 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 method is used in cell biology and for microscopy of intact, often chemically cleared, organs, embryos, and organisms.

Light sheet fluorescence microscopy combines good z-sectioning (as confocal) and illuminates only the observed plane 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.Ĭomparison of different microscopy illumination modalities (LSFM: light sheet fluorescence microscopy, WF: widefield microscopy, CF: confocal microscopy). Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. As only the actually observed section is illuminated, this method reduces the photodamage and stress induced on a living sample.

A second method uses a circular beam scanned in one direction to create the lightsheet. a laser beam which is focused only in one direction (e.g. For illumination, a laser light-sheet is used, i.e.

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. 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. The principle setup of a light sheet fluorescence microscope.

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