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Chintan Patel, Rick Torres, Eben Olson, Michael Levene, Matthew S Lawrence; Multiphoton microscopy for three dimensional histology of retinal whole mounts. Invest. Ophthalmol. Vis. Sci. 2019;60(9):183.
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© ARVO (1962-2015); The Authors (2016-present)
We have established a multiphoton microscopy method optimized for pathology evaluation of whole retinal tissue ex vivo. Standard retina histology methods typically result in incomplete sampling with fragmented sectioning, making overall architectural characterization difficult and rendering three-dimensional (3D) visualization essentially impossible. Clearing Histology with Multiphoton Microscopy (CHiMP) employs chemical clearing and a customized high speed microscope for high resolution imaging at depth, capable of producing 3D reconstructions of entire specimens in a few hours. In this study we apply these newly developed tools to analyze injected green fluorescent protein vector distribution patterns and present complete 3D retinal histopathology reconstructions that reproduce standard histology staining.
African green monkeys eyes were fixed in 4% paraformaldehyde. Retinal whole mounts were cut radially at 4 quadrants for flattening and cleared by serial 2-hour dehydration in 75% ethanol and 100% ethanol, followed by overnight immersion in ethyl cinnamate or benzyl alcohol/benzyl benzoate mixture. Nuclear (DAPI) and protein (Eosin Y) contrast dyes were incubated during dehydration steps. Samples were imaged on a custom multiphoton microscope based on a Ti-Sapphire laser with polygonal and stage scanning and a long working distance index-matched objective.
Retinal flatmounts were imaged at a depth of up to 1 mm to account for sample irregularities. Pseudo-colored high-resolution images revealed distinct cell types including endothelial cells, retinal ganglion cells, photoreceptors and RPE cells in respective retinal layers. Comparison of 3D reconstructions of normal vs. gliotoxin-induced retinal degeneration tissue revealed fine details including numerous fibrotic tangles at the displaced nerve fiber layer, red blood cells in hemorrhagic areas, marked changes in distribution of nuclei and distortion of retinal layers. In another application, a wide distribution of AAV-mediated green fluorescent protein (GFP) expression was visualized to allow localization and quantification of GFP-positive cells.
CHiMP analysis of retinal whole mounts allows visualization perspectives and throughput impossible to achieve with traditional histologic techniques, improving characterization of tissue injury, cell abundance within tissue layers, and vector distribution.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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