Consistent Automatic Spectral Signature Recovery of Human retinal pigment epithelium (RPE) Lipofuscin Components and Drusen in Donors with Age-related Macular Degeneration (AMD) using Multi-Excitation Hyperspectral Autofluorescence (AF) Imaging.

Abstract

Purpose : Three main AF spectral signatures of normal human RPE lipofuscin (LF) S1, S2 and melanolipofuscin (MLF) S3, have been described ex vivo (PMID: 27226929), and in patients with AMD, a sensitive and specific non-LF spectral drusen signature SDr (PMID: 277496960). Prior strategies employed user guided algorithms. We characterize the spectral signatures of these yet to be determined families of fluorophore using a robust, unbiased fully automatic spectral decomposition method with 4 excitation wavelengths.

Methods : Seven RPE flat mounts from seven AMD donors underwent 40X field hyperspectral AF imaging at four excitation wavelengths: 436, 480, 505 and 555 nm, with emissions recorded at 10 nm intervals, 420-720 nm. The 4 hyperspectral data sets for each tissue underwent simultaneous blind source separation into spectra and abundance source images using non-negative matrix factorization initialized by the first 4 non-negative singular-triplets from singular value decomposition [Boutsidis et al, Pattern Recognition, 2008].

Results : Four distinct and characteristic AF emission spectra, 3 from RPE and one from drusen, of human AMD donors were recovered from each of seven tissues. For each of the four spectra, the corresponding spectra from each tissue showed consistently reproducible peaks, shapes and abundance images (Fig 1), across all 4 excitations (Fig 2).

Conclusions : Consistent, automated and unbiased spectral signature recovery across 7 RPE tissues support the physical existence of currently unknown human fluorophore families with these signatures that should be identified with imaging mass spectroscopy for better understanding of RPE physiology in health and disease.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

Figure 1. Spectra and tissue abundances of human RPE lipofuscin and drusen. Top Row: AF micrographs, 3 RPE flatmounts. Second Row: 4 similar AF spectra (436 nm excitation) from each tissue. Next four rows: Corresponding tissue abundances. Spectrum 2, SDr, localizes to drusen and subRPE deposits. The other spectra localize to the LF compartment.

 

Figure 2. Progression of spectra across excitations. RPE spectrum S1 (top) and Drusen spectrum SDr (bottom) from 3 tissues excited at 436, 480, 505 and 555 nm with consistent shapes and peaks.