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Open-Top Light Sheet Microscope

The Open-Top Light-Sheet (OTLS) microscope is tailored to the needs of pathology practice and has been installed in the Botnar Research Centre (May 2023). The system creates pseudo-H&Es with small-molecule fast-acting nuclear (TO-PRO3) and cytoplasmic (Eosin) fluorescent dyes to aid with prognosis.

The instrument will mainly be involved in the investigation of features of lethality and non-lethality in prostate cancer tissue at diagnosis, using light sheet three-dimensional (3D) microscopy with ProtecT diagnostic samples (Prostate Testing for Cancer and Treatment – Prostate Cancer UK).

The hybrid microscope consists of 3 objectives located below the specimen. The illumination objective is combined with two imaging objectives for orthogonal dual-objective (ODO) and non-orthogonal dual-objective (NODO) imaging.

The OTLS design achieves an impressive balance of performance and versatility in a single system for a growing number of cleared-tissue imaging techniques in which high-resolution characterization of localized subregions is desired.

The OTLS system, developed by Jonathan Liu's lab at the University of Washington, is installed with the non-orthogonal dual-objective (NODO), allowing for high resolution of large tissue samples/organs.

Using the NODO imaging path, the system can be tuned to have a lateral resolution of between 400 - 800 nm and an axial resolution of ~2.5 microns. The objectives are positioned below the specimen, which provides an unobstructed open top that enables 3D imaging over a large 12 × 7.5 × 1 cm (xyz) imaging volume (see image left).

The imaging volume is limited in z by the objectives and in xy by the mechanical limits of the motorized stage. The objectives are sealed into a monolithic imaging chamber through direct immersion or the use of a solid-immersion meniscus lens (SIMlens), which provides multi-immersion capabilities spanning the refractive index range of all current clearing protocols.

The system has been used with ECi, PEGASOS, SHIELD, Ce3D, iDISCO, ClearSee/DEEP-Clear and CUBIC clearing techniques. This allows for flexible clearing protocols depending on the type of tissue sample or animal model.

Please enquire about system access/use with the DPOC facility team.

 

Image: The hybrid microscope consists of 3 objectives located below the specimen. The illumination objective is combined with two imaging objectives for orthogonal dual-objective (ODO) and non-orthogonal dual-objective (NODO) imaging. Glaser et al. 2022.

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