title: Spatial and Spectral Characterization of Human Retinal Pigment Epithelium Fluorophore Families by Ex Vivo Hyperspectral Autofluorescence Imaging.
abstract: OBJECTIVE
  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 atλex436 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). Atλex480 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.
  
  
  CONCLUSIONS
  Our results help establish hyperspectral autofluorescence imaging of the human retinal pigment epithelium as a useful tool for investigating retinal health and disease.
authors.affiliation: Department of Ophthalmology, New York University School of Medicine, New York, New York, USA.
authors.forename: Tal
authors.initials: T
authors.lastname: Ben Ami
authors.affiliation: Department of Ophthalmology, New York University School of Medicine, New York, New York, USA.
authors.forename: Yuehong
authors.initials: Y
authors.lastname: Tong
authors.affiliation: Department of Ophthalmology, New York University School of Medicine, New York, New York, USA.
authors.forename: Alauddin
authors.initials: A
authors.lastname: Bhuiyan
authors.affiliation: Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Carrie
authors.initials: C
authors.lastname: Huisingh
authors.affiliation: Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, USA.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Ablonczy
authors.affiliation: Department of Ophthalmology, University Hospital of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ach
authors.affiliation: Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Curcio
authors.affiliation: Department of Ophthalmology, New York University School of Medicine, New York, New York, USA.
authors.forename: R Theodore
authors.initials: RT
authors.lastname: Smith
keywords: autofluorescence imaging; bisretinoids; hyperspectral imaging; lipofuscin; retinal pigment epithelium