Improved spectral recovery and tissue localization of autofluorescence (AF) signatures in donor eyes with age-related macular degeneration (AMD) using multi excitation hyperspectral AF imaging.

Abstract

Purpose : In patients with AMD, clinical fundus AF imaging reveals important pathologic changes. We previously characterized these fluorophore signatures ex vivo using hyperspectral AF imaging with two excitation wavelengths, 436 and 480 nm (Ben Ami et al., PMID 27226929; Tong et al., PMID: 27749696). We extend this work with ex vivo multi-excitation hyperspectral data analysis, to improve identification of retinal pigment epithelium (RPE) and drusen fluorophore signatures.

Methods : Seven RPE flat mounts from 7 donors with AMD were used for hyperspectral AF imaging at 3 excitation wavelengths: 436, 480, and 505 nm. Spectra were then simultaneously decomposed using nonnegative matrix factorization (NNMF) into multiple emission spectra and their corresponding constitutive tissue localizations. Results were compared to those recovered from excitations at 436 and 480 nm only.

Results : Three-wavelength excitation AF provides more detailed and less noisy spectral decompositions of the previously describedmain spectra of RPE fluorophores (S1, S2, S3) and SDrusen (SDr) (Ben Ami et al., PMID 27226929, Tong et al., PMID: 27749696) (Figure 1). In figure 2, we see abundance images that disclose significantly improved tissue localization detail, especially for druse interiors.

Conclusions : Multi-excitatory AF imaging using 3 wavelengths provides richer data for NNMF than does 2-wavelength excitation, with improved tissue detail of drusen and sub RPE deposits. These findings could have implications for specifications of a clinical hyperspectral camera.

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

 

Figure 1. The emission spectra for four fluorophore (S1-S3 and SDr) family components for each of the three excitatory wavelengths. S0 not shown.

 

Figure 2. (left) Tissue abundances were recovered with excitatory wavelengths 436 and 480 nm and (right) tissue abundances of the same spectra were recovered with three excitatory wavelengths, 436, 480 and 505 nm. The upper images are of drusen with effaced RPE overlying the apex and halos of brightness attributable to thickened RPE surrounding the druse base. The lower panels show abundance images for the spectrum of drusen. Note significantly increased detail in images on the right, especially for the internal structure of drusen.