June 2013
Volume 54, Issue 15
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ARVO Annual Meeting Abstract  |   June 2013
Tissue, microscopy, and computational techniques for mapping human retinal pigment epithelium cell number and autofluorescence
Thomas Ach; Jeffrey Messinger; Mark Bentley; Francois Delori; Kenneth Sloan; Christine Curcio
Author Affiliations & Notes
  • Thomas Ach
    Dept of Ophthalmology, University of Alabama at Birmingham, Birmingham, AL
  • Jeffrey Messinger
    Dept of Ophthalmology, University of Alabama at Birmingham, Birmingham, AL
  • Mark Bentley
    Computer and Information Science, University of Alabama at Birmingham, Birmingham, AL
  • Francois Delori
    Ophthalmology, Schepens Eye Research Institute, Boston, MA
  • Kenneth Sloan
    Computer and Information Science, University of Alabama at Birmingham, Birmingham, AL
  • Christine Curcio
    Dept of Ophthalmology, University of Alabama at Birmingham, Birmingham, AL
  • Footnotes
    Commercial Relationships Thomas Ach, None; Jeffrey Messinger, None; Mark Bentley, None; Francois Delori, None; Kenneth Sloan, None; Christine Curcio, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5497. doi:
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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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Abstract
 
Purpose
 

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.

 
Methods
 

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.

 
Results
 

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.

 
Conclusions
 

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.

 
Projection image of a multiplane z-stack from an RPE-only flat-mount (human donor, 83 years). The phalloidin RPE cytoskeleton stain delineates single RPE cells. Autofluorescent granules within RPE cells (exc. 460-490nm) are detected and recorded for further evaluation.
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Projection image of a multiplane z-stack from an RPE-only flat-mount (human donor, 83 years). The phalloidin RPE cytoskeleton stain delineates single RPE cells. Autofluorescent granules within RPE cells (exc. 460-490nm) are detected and recorded for further evaluation.
 
Projection image of a multiplane z-stack from an RPE-only flat-mount (human donor, 83 years). The phalloidin RPE cytoskeleton stain delineates single RPE cells. Autofluorescent granules within RPE cells (exc. 460-490nm) are detected and recorded for further evaluation.
Projection image of a multiplane z-stack from an RPE-only flat-mount (human donor, 83 years). The phalloidin RPE cytoskeleton stain delineates single RPE cells. Autofluorescent granules within RPE cells (exc. 460-490nm) are detected and recorded for further evaluation.
View OriginalDownload Slide
 
Keywords: 701 retinal pigment epithelium • 551 imaging/image analysis: non-clinical • 599 microscopy: light/fluorescence/immunohistochemistry  
© 2013, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2015 Association for Research in Vision and Ophthalmology.
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