SPOT (SPIM-OPT)

Statistical Projection Optical Tomography

SPOT (SPIM-OPT)

SPIM

Selective Plane Illumination Microscopy (SPIM) provides volumetric images of small specimens (between 0.5-10 mm, depending on their transparency). SPIM is based on having the illumination axis and the detection axis orthogonal to each other. SPIM shapes illumination as a thin plane of light (light sheet) orthogonal to the detection axis; therefore the sample is illuminated from the side in a well-defined volume around the focal plane of the detection optics. As only a slice of the sample is illuminated, optical sectioning is effectively performed and fluorescence bleaching is diminished. The combination of various laser sources and different emission filters for detection, makes SPIM ideal for multispectral imaging of emitting regions inside biological samples.

OPT

OPT has emerged as a powerful tool for 3D visualization of specimens across 1–10mm in size, combining fluorescence and absorption imaging. The technique records projections perpendicular to the rotational axis of the specimen from multiple equidistant angles. Standard filtered back projection algorithms of each angle are used for the reconstruction of all slices, producing 3D volumetric data of the specimen. OPT is mostly used on fixed specimens after optical clearing in order to reduce photon scattering, or small live embryos.

SPIM vs OPT

Although both techniques provide spatial resolution at the micrometer scale for mesoscopic (millimeter to centimeter) samples, LSFM provides higher lateral resolution than OPT (∼3 μm vs 8.75 μm) but lower axial (∼25.8 μm vs ∼8.75 μm) for a similar sample (∼2 mm). Moreover, OPT provides isotropic spatial resolution due to its rotational scanning which may reduce the ambiguity in 3D analysis, so it is more adequate for relatively large samples*. OPT has the advantage of recording non fluorescent features, while SPIM has the advantage of being rather faster since there is no need for data reconstruction.

*Chen, et all. (2019), Journal of Microscopy, 275: 3-10

SPOT (SPIM-OPT)

QLS-scope is based on the patented technology called Statistical Projection Optical Tomography (SPOT), that essentially combines two different microscopy approaches (SPIM and OPT) in one system. The benefits of such combination are already mentioned in several research works*,**. QLS-scope is the only commercial system that uses such technology and can provide high lateral and axial resolution, as well as true isotropic images.

*Ripoll, et. all, Optics Express, 2013 | Vol. 21, No. 22 | 25913

**Swoger, et all, Optics Letters, February 15, 2014 / Vol. 39, No. 4 / 1053