Hyperspectral Autofluorescence (AF) of Melanin-containing Organelles in Human Retinal Pigment Epithelium (RPE) with Late Age-related Macular Degeneration (AMD)

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).