Ex Vivo Hyperspectral Autofluorescence (AF) Imaging of Retinal Pigment Epithelium (RPE) in Human Donor Eyes with Age-related Macular Degeneration (AMD)

T. B. Ami, Y. Tong, Paul Sajda, Z. Ablonczy, C. Curcio, T. Smith, T. Ach


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.


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.


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).


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.

1. Smith, Post, Johri, Lee, Ablonczy, Curcio, Ach, Sajda: Hyperspectral signal recovery of unknown fluorophores in the RPE. Biomed Opt Express 2014.
2. Ach, ARVO 2014

Accepted 11 June 2015
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