# A hyperspectral approach to RPE fluorophores in AMD

> **NIH NIH R01** · UNIVERSITY OF ALABAMA AT BIRMINGHAM · 2020 · $321,953

## Abstract

Abstract
Age-related macular degeneration (AMD) causes vision loss in millions worldwide. Central to AMD initiation
and progression is the retinal pigment epithelium (RPE), which is clinically visualized via the massed
autofluorescence (AF) of its lipofuscin and melanolipofuscin granules. We hypothesize, based on our imaging
and pathology studies, that AMD can be staged and monitored by the expression and distribution of unique
RPE fluorophores. We identified and characterized ex vivo spectral signatures of distinct fluorophore families
in normal RPE, Bruch's membrane and drusen, AMD's hallmark lesion. However, the molecular sources of
these spectra in the macula are now uncertain due to seminal imaging mass spectrometry (IMS) studies
showing that a major lipofuscin fluorophore, A2E, is abundant in the periphery. Thus, the long-term goal of
this 6-investigator collaboration is to develop AMD diagnostics based on hyperspectral AF for spectral,
molecular biopsy of the RPE, linking clinical pathology to underlying molecular composition. Hyperspectral
AF imaging, unlike conventional AF imaging, acquires 3-dimensional “hypercubes” of data (2 spatial
coordinates – x, y - and 1 spectral - wavelength). We explored imaging data with novel tensor-based tools
exploiting multiple excitation wavelengths to discover RPE spectral signatures and their spatial distributions.
We propose to link fluorophores to granules, RPE cells, tissue, and AMD stages in 3 aims using a common
human tissue source. Aim 1 uses hyperspectral AF tissue mapping to understand AMD pathology at the
spectral level in eyes with AMD and unaffected control eyes. We will map RPE flat-mounts by hyperspectral
AF microscopy linked to a pathology grading system. Spectral AF components will be recovered
mathematically and assigned to subcellular and extracellular features. Aim 2 will quantify AMD pathology at
the subcellular level by enumerating fluorophore-containing granules using structured illumination microscopy
and 3-dimensional electron microscopy. Aim 3 uses hyperspectral fluorophore identification to understand
AMD pathology at the molecular level. We will determine candidate molecules for the major spectral
components discovered in Aim 1 by hyperspectral thin layer chromatography and will verify their spatial
distributions by IMS. Synthetic authentic standards will ensure spectral validation. Biological validation at this
level is unprecedented for clinical ophthalmology, yet warranted by the size of the AMD patient population,
the enormity of knowledge gap about major RPE fluorophores in human eyes, the availability of donor tissue,
and the proven success of validating other imaging technologies. Our results will directly translate to clinical
hyperspectral AF imaging for noninvasive, spatially precise early detection and longitudinal AMD follow-up, in
vivo target discovery, and immediate extensions beyond AMD. From our discoveries will flow a huge range of
experiments in outer retinal ...

## Key facts

- **NIH application ID:** 9965926
- **Project number:** 5R01EY027948-04
- **Recipient organization:** UNIVERSITY OF ALABAMA AT BIRMINGHAM
- **Principal Investigator:** Thomas Ach
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $321,953
- **Award type:** 5
- **Project period:** 2017-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9965926

## Citation

> US National Institutes of Health, RePORTER application 9965926, A hyperspectral approach to RPE fluorophores in AMD (5R01EY027948-04). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9965926. Licensed CC0.

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