Characterizing Spectra from Hyperspectral Autofluorescence (AF) of Human Eyes with and without Age-related Macular Degeneration (AMD)

Abstract

Purpose : Hyperspectral AF imaging with non-negative matrix factorization (NMF) produces spectra of physiologic and pathologic human RPE fluorophores in AMD (Tong et al., PMID 27749696). Characterizing these spectra can deepen understanding of AMD and offer clinical prognostic value.

Methods : RPE flatmounts of 19 eyes, from 9 patients with AMD or probable AMD and 10 without, were captured at 40X at the fovea, perifovea, periphery, and areas with drusen for a total of 68 images. AF excitation occurred at 436, 480, and 505 nm. Emissions were collected from 420 to 720 nm, decomposed by NMF to five spectra and abundances, normalized, grouped by peak emission, and analyzed for peak width and secondary peaks. Peak width is defined as the distance between half of max surrounding the peak emission.

Results : In 340 total spectra, 315 were nonzero with peak emissions from 436 nm excitation at 420, 490, 510, 520, 550, 580, and 620 nm. The peak at 420 nm likely represents artifact. A graph of all spectra that peak at 550 nm is attached (Fig. 1) as an example of a group with high intragroup similarity based on further characterization. Outliers were removed from further analysis in 4/6 peak groups.
Intragroup peak width had a standard deviation less than 35 nm, with mean peak widths reported below (Fig. 2). The groups with peaks at 520, 550, 580, and 620 nm had secondary peaks at 600, 610, 610, and 720 nm respectively, and over 84% of each intragroup spectra had the same secondary peak, with all percentages reported (Fig. 3).

Conclusions : NMF spectra from 3 wavelength excitation grouped by peak emission represent previously described fluorophores from excitation at 2 wavelengths: S1A peaking at 520 nm, S1B at 550 nm, S2 at 580 nm, S3 at 620 nm, Bruch’s Membrane at 490 nm, and drusen, found only in AMD eyes, at 510 nm (Ben Ami et al., PMID 27226929). Exemplary spectra show intragroup similarity in peak width and secondary peaks which can aid in identifying their molecular sources.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Figure 1. A plot of all exemplary spectra peaking at 550 nm. The first peak shows emission from excitation at 436 nm, the second at 480 nm, and the third at 505 nm.

 

Figure 2. Above: The mean peak width of each of the spectra when grouped by peak emission wavelength, with peak width defined above. Error bars represent standard deviation. Below: The percentage of intragroup spectra with a shared secondary peak.