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Thomas Ach, Jeffrey Messinger, Mark Bentley, Francois Delori, Kenneth Sloan, Christine Curcio; Tissue, microscopy, and computational techniques for mapping human retinal pigment epithelium cell number and autofluorescence. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5497.
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© ARVO (1962-2015); The Authors (2016-present)
To describe methods for systematic examination of retinal pigment epithelium (RPE) flat-mounts for quantifying cell number counts and total autofluorescence (AF) as a function of position relative to the fovea in eyes of different ages and maculopathy status.
Belts of chorioretinal tissue from human donor eyes fixed in paraformaldehyde were previously cryo-preserved in glycerol/PBS buffer and frozen. RPE flat-mounts from these specimens were prepared after thawing and rehydration. Different stages of tissue preparation were photodocumented: retina on, retina off, and retina+choroid off. RPE-only flat-mounts were labeled with AlexaFluor647-phalloidin for filamentous actin and imaged on a spinning disk confocal fluorescence microscope. Multiplane z-series of RPE-only flat-mounts for AF measurements (exc. 460-490nm, em. > 505nm) were collected and evaluated with the integrated software in relation to a fluorescence reference now available (Delori et al., PMID 22016060). An ImageJ plug-in counted RPE cell bodies delimited by phalloidin-labeled cytoskeleton, with user-editing capabilities.
Removing the retina and choroid in preparing RPE-only flat-mounts is challenging but necessary for obtaining a smooth tissue for accurate microscopy. An image overlay showing landmarks common to the different dissection steps specifies the exact location of the fovea. From these landmarks, locations on the RPE-only flat-mounts can be specified. Phalloidin delineates RPE cell borders (Figure) and is suitable for reproducible cell number counts performed with the custom software. AF properties of the counted RPE cells can then be detected (Figure). Normalization to a fluorescence reference allows comparison of RPE cell AF characteristics at different locations.
The RPE-only flat-mount tissue preparation, microscopy, and computational methods allow an accurate, reproducible mapping of RPE cell number and AF properties at clearly defined locations. These combined techniques will be the basis for further systematic analysis and comparison of RPE flat-mounts from healthy eyes and eyes with AMD.
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