Ben Ami Tal, Tong Yuehong, Bhuiyan Alauddin, Huisingh Carrie, Ablonczy Zsolt, Ach Thomas, Curcio Christine A, Smith R Theodore
Department of Ophthalmology, New York University School of Medicine, New York, New York, USA.
Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Transl Vis Sci Technol. 2016 May 17;5(3):5. doi: 10.1167/tvst.5.3.5. eCollection 2016 May.
Discovery of candidate spectra for abundant fluorophore families in human retinal pigment epithelium (RPE) by ex vivo hyperspectral imaging.
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.
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 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 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.
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.
Our results help establish hyperspectral autofluorescence imaging of the human retinal pigment epithelium as a useful tool for investigating retinal health and disease.
通过离体高光谱成像发现人视网膜色素上皮（RPE）中丰富荧光团家族的候选光谱。
在20个正常RPE/布鲁赫膜（BrM）平铺标本的三个位置（中央凹、中央凹周围、近周边），于两个激发波段（436 - 460、480 - 510 nm）下，采集420至720 nm（间隔10 nm）的高光谱自发荧光发射图像。数学分解从每个组织中提取出一个BrM光谱（S0）和源自RPE的丰富脂褐素/黑素脂褐素（LF/ML）光谱（S1、S2、S3）。
分析纳入了在所有三个视网膜位置以及14只眼（84个数据集）的两种激发情况下，具有与LF/ML一致的核周定位的平滑光谱S1至S3。在436 nm处，S0、S1和S2的平均峰值发射分别为495±14、535±17和576±20 nm。S3通常为三峰，峰值位于580、620或650 nm（峰值模式为650 nm）。在480 nm处，S0、S1和S2红移至526±9、553±10和588±23 nm，且S3再次为三峰（峰值模式为620 nm）。S1常分裂为两个光谱，即S1A和S1B。S3与黑色素强烈共定位。年龄、性别或视网膜位置之间无显著差异。
在该健康眼样本中，似乎至少存在三个丰富的RPE荧光团家族，它们在年龄、视网膜位置和性别上普遍存在。通过成像质谱进行进一步的分子表征以及通过超分辨率显微镜进行定位，应能阐明涉及荧光团的正常和异常RPE生理学。
我们的结果有助于将人视网膜色素上皮的高光谱自发荧光成像确立为研究视网膜健康和疾病的有用工具。
