Hybrid open-top light sheet microscopy for cleared tissues

This Learning Wednesday paper note highlights “A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.” The study is relevant to hybrid open-top light-sheet microscopy for multiscale cleared-tissue imaging, with a focus on how three-dimensional tissue context can alter what researchers see and measure.

Selected notes from the paper

"Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues.

"There is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution, sample geometry, and compatibility with tissue-clearing protocols and sample holders of various refractive indices."

"We designed a ‘hybrid’ OTLS microscope that is the first system to use a non-orthogonal dual-objective (NODO) configuration (for high-resolution imaging)."

"For low-resolution imaging, a conventional ODO open-top system is integrated with the NODO system."

"The hybrid microscope architecture consists of three objectives, positioned below the specimen, mounted into a monolithic imaging chamber that is filled with an interchangeable immersion medium."

"By using a motorized stage, tiled imaging is possible with both paths over a large 12 x 7.5 x 1 cm (XYZ) imaging volume."

"The ODO path provides fast meso-scale screening capabilities, and the NODO path enables targeted sub-micrometer imaging."

"This large imaging volume accommodates multiple intact cleared organs and large tissue slabs mounted in an array of specimen holders."

"Using the ODO imaging path, a mouse brain was rapidly screened in ~1 hour with isotropic ~2-μm voxels (~4-μm resolution), revealing brain-wide axonal projections."

"A targeted region of interest around a cortical pyramidal neuron was then imaged at <0.5-um xy resolution using the NODO imaging path."

"This imaging resolution is sufficient to discern spines and varicosities on individual dendrites and axons."

"We used our hybrid OTLS system to study metastatic colonies from two cancer cell lines (MDA-231 and OS-RC-2) throughout intact mouse brains."

"While past studies have relied on laborious and time-consuming experiments with manual transfer of individual specimens between different microscope systems, we analyzed six intact mouse brains in a single imaging session without manually removing or remounting the specimens."

"Our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications."

From an Alpenglow perspective, this paper is useful because it connects hybrid open-top light-sheet microscopy for multiscale cleared-tissue imaging to a broader need in 3D spatial biology: measuring tissue architecture across depth while preserving context for quantitative analysis.