TY  - JOUR
A1  - Ben Ami, Tal
A1  - Tong, Yuehong
A1  - Bhuiyan, Alauddin
A1  - Huisingh, Carrie
A1  - Ablonczy, Zsolt
A1  - Ach, Thomas
A1  - Curcio, Christine A.
A1  - Smith, R. Theodore
T1  - Spatial and Spectral Characterization of Human Retinal Pigment Epithelium Fluorophore Families by Ex Vivo Hyperspectral Autofluorescence Imaging
JF  - Translational Vision Science & Technology
N2  - Purpose: 
Discovery of candidate spectra for abundant fluorophore families in human retinal pigment epithelium (RPE) by ex vivo hyperspectral imaging.

Methods: 
Hyperspectral autofluorescence emission images were captured between 420 and 720 nm (10-nm intervals), at two excitation bands (436–460, 480–510 nm), from three locations (fovea, perifovea, near-periphery) in 20 normal RPE/Bruch's membrane (BrM) flatmounts. Mathematical factorization extracted a BrM spectrum (S0) and abundant lipofuscin/melanolipofuscin (LF/ML) spectra of RPE origin (S1, S2, S3) from each tissue.

Results: 
Smooth spectra S1 to S3, with perinuclear localization consistent with LF/ML at all three retinal locations and both excitations in 14 eyes (84 datasets), were included in the analysis. The mean peak emissions of S0, S1, and S2 at λ\(_{ex}\) 436 nm were, respectively, 495 ± 14, 535 ± 17, and 576 ± 20 nm. S3 was generally trimodal, with peaks at either 580, 620, or 650 nm (peak mode, 650 nm). At λ\(_{ex}\) 480 nm, S0, S1, and S2 were red-shifted to 526 ± 9, 553 ± 10, and 588 ± 23 nm, and S3 was again trimodal (peak mode, 620 nm). S1 often split into two spectra, S1A and S1B. S3 strongly colocalized with melanin. There were no significant differences across age, sex, or retinal location.

Conclusions: 
There appear to be at least three families of abundant RPE fluorophores that are ubiquitous across age, retinal location, and sex in this sample of healthy eyes. Further molecular characterization by imaging mass spectrometry and localization via super-resolution microscopy should elucidate normal and abnormal RPE physiology involving fluorophores.

Translational Relevance: 
Our results help establish hyperspectral autofluorescence imaging of the human retinal pigment epithelium as a useful tool for investigating retinal health and disease.
KW  - spectral characterization
KW  - human retinal pigment epithelium
KW  - fluorophores
KW  - hyperspectral autofluorescence imaging
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-168328
VL  - 5
IS  - 3
ER  -