June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Ex Vivo Hyperspectral Autofluorescence (AF) Imaging of Retinal Pigment Epithelium (RPE) in Human Donor Eyes with Age-related Macular Degeneration (AMD)
Author Affiliations & Notes
  • Tal Ben Ami
    Ophthalmology, New York University School of Medicine, New York, NY
  • Yuehong Tong
    Ophthalmology, New York University School of Medicine, New York, NY
  • Paul Sajda
    Biomedical Engineering, Columbia University, New York, NY
  • Zsolt Ablonczy
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Christine A Curcio
    Ophthalmology, University of Alabama at Birmingham School of Medicine, Birmingham, AL
  • Theodore Smith
    Ophthalmology, New York University School of Medicine, New York, NY
  • Thomas Ach
    Ophthalmology, University of Alabama at Birmingham School of Medicine, Birmingham, AL
  • Footnotes
    Commercial Relationships Tal Ben Ami, None; Yuehong Tong, None; Paul Sajda, None; Zsolt Ablonczy, None; Christine Curcio, None; Theodore Smith, Advanced Cell Technologies (C); Thomas Ach, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 4369. doi:https://doi.org/
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      Tal Ben Ami, Yuehong Tong, Paul Sajda, Zsolt Ablonczy, Christine A Curcio, Theodore Smith, Thomas Ach; Ex Vivo Hyperspectral Autofluorescence (AF) Imaging of Retinal Pigment Epithelium (RPE) in Human Donor Eyes with Age-related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4369. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

We have shown how hyperspectral imaging can extract individual signals of abundant fluorophores ex-vivo in normal RPE [1]. Herein we analyze donor eyes with AMD, hypothesizing that the spectral signatures would vary with perturbations in RPE physiology.

 
Methods
 

Hyperspectral AF images were captured from 7 locations in 5 RPE/Bruch’s-membrane (BrM) flat-mounts from donor eyes with early to late AMD. Imaging was performed at 2 excitation bands, 436-460 and 480-510 nm, with emission captured between 420 and 720 nm in 10 nm intervals using a Nuance FX camera.

 
Results
 

Gaussian mixture modeling and mathematical factorization were applied to extract 1 BrM spectrum and 4 abundant emission spectra from RPE organelles, the latter peaking at mean wavelengths of 519±7, 574±8, 599±4, and 644±10 nm (436 nm excitation). The 519 nm peak was blue-shifted ~50 nm relative to the corresponding signal from normal eyes [1]. Spatial abundance images showed unique signals localized to RPE granule aggregates, melanosomes, and basal laminar deposits (BLamD) (Figure).

 
Conclusions
 

In AMD, some AF signals from RPE become spatially discrete [2], and a morphological diversity of RPE granule populations is well demonstrated using hyperspectral imaging. Differences in spectra and their spatial distributions between normal and AMD eyes may extend our knowledge of RPE pathophysiology in AMD.<br /> <br /> 1. Smith, Post, Johri, Lee, Ablonczy, Curcio, Ach, Sajda: Hyperspectral signal recovery of unknown fluorophores in the RPE. Biomed Opt Express 2014.<br /> 2. Ach, ARVO 2014  

 
Hyperspectral images of an eye with geographic atrophy from an 81-year-old donor. RGB panel: Composite RGB AF image of an atrophic area containing RPE cells (yellow) overlying BrM (green), showing 2 RPE cells with prominent signal (gray arrows), granule aggregates (white arrows), sub-RPE tissue including BrM and persistent BLamD with brighter but cloudy, non-uniform AF (pink arrows). Graph: The 5 spectra (C1 to C5) recovered at 436 nm excitation, and their spatial abundance images (C2-C4 shown, warm colors indicate greater abundance). C2 represents persistent BLamD blocked by melanosomes, C3 shows cells and aggregates, and C4 is consistent with melanosomes. Overlay panel: Each component is assigned its own color, with mixing occurring where components co-localize.
 
Hyperspectral images of an eye with geographic atrophy from an 81-year-old donor. RGB panel: Composite RGB AF image of an atrophic area containing RPE cells (yellow) overlying BrM (green), showing 2 RPE cells with prominent signal (gray arrows), granule aggregates (white arrows), sub-RPE tissue including BrM and persistent BLamD with brighter but cloudy, non-uniform AF (pink arrows). Graph: The 5 spectra (C1 to C5) recovered at 436 nm excitation, and their spatial abundance images (C2-C4 shown, warm colors indicate greater abundance). C2 represents persistent BLamD blocked by melanosomes, C3 shows cells and aggregates, and C4 is consistent with melanosomes. Overlay panel: Each component is assigned its own color, with mixing occurring where components co-localize.

 
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