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Open AccessArticle

Ex Vivo Hyperspectral Autofluorescence Imaging and Localization of Fluorophores in Human Eyes with Age-Related Macular Degeneration

1
Department of Ophthalmology, New York University School of Medicine, New York, NY 10016, USA
2
Department of Ophthalmology, Icahn School of Medicine of Mount Sinai, New York, NY 10029, USA
3
Leibniz Institute of Photonic Technology, 07745 Jena, Germany
4
Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, 07743 Jena, Germany
5
Department of Ophthalmology, University Hospital Jena, 07743 Jena, Germany
6
Center for Medical Optics and Photonics, University of Jena, 07743 Jena, Germany
7
Department of Ophthalmology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35233, USA
8
Department of Ophthalmology, University Hospital of Würzburg, 97080 Würzburg, Germany
9
Department of Ophthalmology, Medical University of South Carolina, Charleston, SC 29425, USA
*
Author to whom correspondence should be addressed.
Vision 2018, 2(4), 38; https://doi.org/10.3390/vision2040038
Received: 2 April 2018 / Revised: 19 May 2018 / Accepted: 12 September 2018 / Published: 26 September 2018
(This article belongs to the Special Issue Age-Related Macular Degeneration)
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. View Full-Text
Keywords: age-related macular degeneration; autofluorescence; drusen; non-negative matrix factorization; hyperspectral imaging; retinal pigment epithelium age-related macular degeneration; autofluorescence; drusen; non-negative matrix factorization; hyperspectral imaging; retinal pigment epithelium
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Mohammed, T.; Tong, Y.; Agee, J.; Challa, N.; Heintzmann, R.; Hammer, M.; Curcio, C.A.; Ach, T.; Ablonczy, Z.; Smith, R.T. Ex Vivo Hyperspectral Autofluorescence Imaging and Localization of Fluorophores in Human Eyes with Age-Related Macular Degeneration. Vision 2018, 2, 38.

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