lightsheet

Light sheet, in the context of microscopy, refers to light-sheet fluorescence microscopy (LSFM), a technique that illuminates a specimen with a thin sheet of light while collecting emitted fluorescence with a detector oriented orthogonally to the illumination. This arrangement excites only the plane being imaged, providing optical sectioning with reduced photobleaching and phototoxicity compared with traditional wide-field or confocal methods.

In a typical LSFM setup, a laser is shaped into a narrow sheet of light using a cylindrical lens or by scanning a focused beam to form a sheet. The detection objective, positioned perpendicular to the sheet, captures fluorescence from the illuminated plane. By moving the sheet (or the sample) through the volume, a three-dimensional image stack is constructed. Some implementations use two opposing objectives to improve resolution along axial directions, and various approaches exist to enhance isotropy and speed, such as dual-view SPIM or lattice light-sheet variants.

History and terminology: LSFM emerged in the early 2000s and is commonly referred to as single-plane illumination microscopy (SPIM). Since then, it has spawned multiple variants, including diSPIM (dual-view SPIM) and lattice light-sheet microscopy (LLSM), which aims for higher axial resolution and gentler illumination.

Applications: LSFM is especially suited for live imaging of large, opaque or whole organisms and thick tissues, including embryos, zebrafish, Drosophila, and organoids. It enables long-term, fast volumetric imaging with reduced phototoxicity, as well as imaging cleared specimens for deep, high-contrast visualization.

Limitations and considerations: The technique requires careful optical alignment, appropriate sample mounting, and often transparent or optically cleared specimens. It can be specialized and costly, and performance depends on sample scattering, refractive index matching, and the geometry of the illumination and detection paths.