Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

Abstract

Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

CytoMAP: a spatial analysis toolbox reveals features of myeloid cell organization in lymphoid tissues

“…These approaches are providing an unprecedented level of detail into biological processes, and the development of better tools for analyzing increasingly complex and voluminous datasets is critical. 4,10,13,14,22,32,35,39,[53][54][55] Here we developed and described a comprehensive analysis platform, CytoMAP, capable of robust spatial analysis of cellular organization within tissues. CytoMAP harnesses the power of data clustering, dimensionality reduction, and advanced data visualization to expand the utility of spatially resolved cellular profiling.…”

“…histo-cytometry, CODEX, MIBI, STARMAP). 4,[10][11][12][13][14][15][16][17] These techniques generate panoptic datasets describing phenotypic, transcriptional, functional, and morphologic cellular properties, while retaining information on the precise 2-dimensional (2D) or 3D positioning of cells within tissues, thus potentiating a more complete understanding of cellular function directly in situ. Analysis of images generated by these methods requires multiple processing steps, including image preprocessing, cell segmentation, object classification, and spatial analysis.…”

A hybrid open-top light-sheet microscope for multi-scale imaging of cleared tissues

“…We have previously developed a number of open-top light-sheet (OTLS) microscopy systems that provide an ease-of-use similar to that of a flat-bed document scanner, enabling one or more specimens of arbitrary geometry to be imaged without lateral constraints up to a depth of several millimeters [15][16][17]. The OTLS architecture also offers the versatility to accommodate a wide range of potential accessory technologies, such as microfluidics, electrophysiology, and micro-dissection/aspiration.…”

High-Resolution 3D Fluorescent Imaging of Intact Tissues

“…Tissue-clearing protocols that have attempted to address light scattering and penetration limits were recently reviewed 2,3 . While most of these approaches were restricted to embryos and small animal models, three studies demonstrated tissue clearing in human brain or prostate samples [4][5][6] , though it is unknown how these protocols perform in other human organs. The major limitation shared by all previous reports is that the cleared samples were not imageable with high resolution objectives [6][7][8] .…”

“…While most of these approaches were restricted to embryos and small animal models, three studies demonstrated tissue clearing in human brain or prostate samples [4][5][6] , though it is unknown how these protocols perform in other human organs. The major limitation shared by all previous reports is that the cleared samples were not imageable with high resolution objectives [6][7][8] . For the samples to physically fit within the working distance of the objective lens, low powered objectives (i.e.…”