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Abstract
To characterize fluorophore signals from drusen and retinal pigment epithelium (RPE) and their changes in age related macular degeneration (AMD), the authors describe advances in ex vivo hyperspectral autofluorescence (AF) imaging of human eye tissue. Ten RPE flatmounts from eyes with AMD and 10 from eyes without AMD underwent 40× hyperspectral AF microscopic imaging. The number of excitation wavelengths tested was initially two (436 nm and 480 nm), then increased to three (436 nm, 480 nm, and 505 nm). Emission spectra were collected at 10 nm intervals from 420 nm to 720 nm. Non-negative matrix factorization (NMF) algorithms decomposed the hyperspectral images into individual emission spectra and their spatial abundances. These include three distinguishable spectra for RPE fluorophores (S1, S2, and S3) in both AMD and non-AMD eyes, a spectrum for drusen (SDr) only in AMD eyes, and a Bruch’s membrane spectrum that was detectable in normal eyes. Simultaneous analysis of datacubes excited atthree excitation wavelengths revealed more detailed spatial localization of the RPE spectra and SDr within drusen than exciting only at two wavelengths. Within AMD and non-AMD groups, two different NMF initialization methods were tested on each group and converged to qualitatively similar spectra. In AMD, the peaks of the SDr at ~510 nm (436 nm excitation) were particularly consistent. Between AMD and non-AMD groups, corresponding spectra in common, S1, S2, and S3, also had similar peak locations and shapes, but with some differences and further characterization warranted.
Highlights
Deployed in satellite imagery for water resource management [1], hyperspectral imaging has found uses in food safety monitoring [2], forensic medicine [3], and biomedicine [4].By providing spatially resolved spectral imaging, biomedical hyperspectral imaging has demonstrated utility for disease detection [5] and monitoring [6]
Consistent and reproducible recovery of spectral signatures from post‐mortem human retinas support the hypothesis that distinct fluorophores localize to retinal pigment epithelium (RPE) organelles, to Bruch’s membrane, and to drusen [15]
The immediate clinical advantage of this technique, if a camera were available today, is the detection of drusen and especially diffuse deposits of drusen precursors [22] that are not Consistent and reproducible recovery of spectral signatures from post-mortem human retinas support the hypothesis that distinct fluorophores localize to RPE organelles, to Bruch’s membrane, and to drusen [15]
Summary
Deployed in satellite imagery for water resource management [1], hyperspectral imaging has found uses in food safety monitoring [2], forensic medicine [3], and biomedicine [4].By providing spatially resolved spectral imaging, biomedical hyperspectral imaging has demonstrated utility for disease detection [5] and monitoring [6]. Clinical fundus autofluorescence (AF) imaging of the RPE, support cells to photoreceptors, and choroidal vasculature reveals morphologic and pathologic changes in patients with retinal diseases, including age related macular degeneration (AMD) [9,10,11]. This in vivo imaging modality acquires the total AF emission of fluorophores localizing primarily to RPE, as impacted by surrounding tissues, all excited at 488 nm wavelength. This suggests that a clinical HAF camera with similar capabilities, for the drusen spectrum SDr could yield new diagnostic and prognostic information about AMD [15];
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