September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Hyperspectral Autofluorescence (AF) of Melanin-containing Organelles in Human Retinal Pigment Epithelium (RPE) with Late Age-related Macular Degeneration (AMD)
Author Affiliations & Notes
  • Tal Ben Ami
    Ophthalmology, New York University School of Medicine, New York, New York, United States
  • Yuehong Tong
    Ophthalmology, New York University School of Medicine, New York, New York, United States
  • Sungmin Hong
    Computer Science & Engineering, New York University Tandon School Engineering, Brooklyn, New York, United States
  • Rainer Heintzmann
    Institute of Physical Chemistry, Friedrich Schiller University Jena, Jena, Germany
  • Guido Gerig
    Computer Science & Engineering, New York University Tandon School Engineering, Brooklyn, New York, United States
  • Zsolt Ablonczy
    Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, United States
  • Christine A Curcio
    Ophthalmology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
  • Thomas Ach
    Ophthalmology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, United States
    Ophthalmology, University Hospital of Würzburg, Würzburg, Germany
  • Theodore Smith
    Ophthalmology, New York University School of Medicine, New York, New York, United States
  • Footnotes
    Commercial Relationships   Tal Ben Ami, None; Yuehong Tong, None; Sungmin Hong, None; Rainer Heintzmann, None; Guido Gerig, None; Zsolt Ablonczy, None; Christine Curcio, None; Thomas Ach, None; Theodore Smith, None
  • Footnotes
    Support  R01 EY015520 (RTS), R01 EY021470 (RTS), Foundation Fighting Blindness Individual Investigator Research Award (RTS), Research to Prevent Blindness (RTS), NEI EY06109 (CC), 2014 von Sallmann Prize (CC), EyeSight Foundation of Alabama (CC), Research to Prevent Blindness (CC), DFG Ac265/1-1(TA) and DFG Ac265/2-1 (TA), NIH EY-019065 (ZA), An unrestricted grant to the Dept. of Ophthalmology at MUSC from RPB (ZA).
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 1705. doi:
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      Tal Ben Ami, Yuehong Tong, Sungmin Hong, Rainer Heintzmann, Guido Gerig, Zsolt Ablonczy, Christine A Curcio, Thomas Ach, Theodore Smith; Hyperspectral Autofluorescence (AF) of Melanin-containing Organelles in Human Retinal Pigment Epithelium (RPE) with Late Age-related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2016;57(12):1705.

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

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Abstract

Purpose : We have used hyperspectral imaging to extract candidate spectra of fluorophores ex-vivo in normal and diseased RPE (PMID 25574430; ARVO 15' E-Abstracts 3956 & 4369). Herein we characterize a distinct AF signal of melanosomes-melanolipofuscin (M/ML) granules in the RPE of donors with geographic atrophy (GA) and neovascular AMD.

Methods : Hyperspectral AF images were captured at 18 locations from 6 RPE/Bruch’s-membrane flat-mounts of donor eyes with late AMD. Imaging was performed at 2 excitation bands, 436-460 and 480-510 nm; emission was captured between 420-720 nm in 10 nm intervals. Mathematical factorization was applied to extract abundant emission spectra and their spatial abundance images from RPE organelles (labeled S1-S3) and drusen/sub-RPE deposits (labeled SDr). To correlate AF with M/ML, images were compared with those captured by bright-field (BF) microscopy at the same locations.

Results : At 436 nm excitation, a broad AF signal S3 (Fig.1: Spectra & S3) was extracted at all 18 locations, with mean emission maximum of 631±25 nm, range 580-650 nm. The S3 spatial abundance image demonstrated correlation with areas containing M/ML granules in 14/18 locations on BF images (77.8%), and in 12/18 locations on AF composite images (66.7%) (Fig.1: BF & AF). Spectra S1 and S2 showed general correlation with lipofuscin/ML (LF/ML) (Fig.1: S2), not with M/ML granules.

Conclusions : Hyperspectral imaging of the RPE in AMD extracted a consistent AF signal in the red wavelengths that strongly correlated to areas dense with M/ML granules. A similar signal was more diffusely distributed in normal RPE, suggesting a progressive pathophysiology of interest in AMD. Further investigation of melanin distribution in AMD using super-resolution structured illumination and imaging mass spectrometry is warranted.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

86-year-old male donor with GA. BF: Pigmented M/ML granules are seen inside RPE cells at different stages of degeneration. AF: M/ML seen as dark-yellow/brown areas. Spectra: Spectra S1-S3 and SDr recovered at 436 nm excitation. S3 has a broad peak at 630 nm. S3 abundance: demonstrates highest intensity in areas with dense M/ML (arrows). S2 abundance: general correlation with LF/ML granules, but not specifically with M/ML (arrows). SDr abundance: characteristic localization to a central druse and sub-RPE deposits, no correlation with M/ML (arrows).

86-year-old male donor with GA. BF: Pigmented M/ML granules are seen inside RPE cells at different stages of degeneration. AF: M/ML seen as dark-yellow/brown areas. Spectra: Spectra S1-S3 and SDr recovered at 436 nm excitation. S3 has a broad peak at 630 nm. S3 abundance: demonstrates highest intensity in areas with dense M/ML (arrows). S2 abundance: general correlation with LF/ML granules, but not specifically with M/ML (arrows). SDr abundance: characteristic localization to a central druse and sub-RPE deposits, no correlation with M/ML (arrows).

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