Light-Sheet Fluorescence Microscopy

Abstract

In light-sheet fluorescence microscopy (LSFM) the optical axes of the image-forming microscope objective and the objective providing illumination of the specimen are at right angles to one another (Olarte et al., 2018). A sheet of light is incident on the specimen, and fluorescence is produced and hence detected only in that plane. To obtain images of multiple planes for three-dimensional reconstruction the light-sheet is displaced relative to the specimen either by scanning the illumination or by moving the specimen through the light-sheet (Huber et al., 2001). LSFM has the intrinsic capability of optically sectioning the specimen. An LSFM differs markedly from epi-illumination fluorescence microscopes, in which illumination is incident on the entire specimen. The key advantage of LSFM lies in the associated decreased photobleaching and phototoxicity in the specimen—up to three orders of magnitude, depending on the specimen—as compared with an epi-illumination fluorescence microscope (Reynaud et al., 2008).