- Lightsheet, Single Plane Illumination Microscopy (SPIM)

Fluorescence illumination is performed laterally with a laser 'lightsheet', i.e. a plane of light traveling exactly along the image plane, originating from a 90 degrees angle respect to the detection axis. The Lightsheet is static and the sample is scanned through it to perform 3D imaging. The geometry allows for sample rotation, long working distance for large samples, excellent optical sectioning at low NA. Long term Live imaging is one of the major possibility because Phototoxicity is dramatically reduced by the optical geometry: the sample is not illuminated outside of the image plane during acquisition.

Main variations of SPIM (optical architecture) include:

mSPIM (multiple lightsheet from different directions),

muviSPIM: double illumination and double sided detection SPIM (4 objective lenses pointing at the sample),

DSLM (Digitally Scanned Lightsheet Microscopy) where the lightsheet is formed by scanning a 2D-focussed across the image plane, Ultramicroscopy: a specific nomenclature of lightsheet system for cleared sample, usually with upright detection and horizontal ligthsheet propagation.

iSPIM: "Inverted SPIM". Sample mounting like on an inverted microscope; orthogonal light sheet objective lenses on top of the sample, lowered into open chamber with dipping media.

diSPIM: same as iSPIM, but both objective lenses can be used for illumination and detection, enabling one to acquire two orthogonal volumes that, when recombined (post-processing), will increase the resolution to be near isotropic in 3D.

More features:

ASLM, Axially Swept Lightsheet Microscopy: A tightly focused lightsheet is rastered along its propagation axis to distribution the thinnest part (waist) along the entire field of view, image is acquired only where/when the waist travels. Usually Slower than SPIM and some over-exposure, but can be synchronised with cameras rolling shutter. It yields the best compromise between large field of view and high axial resolution.

Bessel beam, Airybeam and/or two-photon: Beam shaping combined with DSLM can provide specific benefits and performance e.g. to improve resolution, penetration, etc...