Autofluorescent Granules of the Human Retinal Pigment Epithelium: Phenotypes, Intracellular Distribution, and Age-Related Topography
Autofluorescent Granules of the Human Retinal Pigment Epithelium: Phenotypes, Intracellular Distribution, and Age-Related Topography
复制标题
DOI：
10.1167/iovs.61.5.35
复制
发表时间：
2020-05-01
影响因子：
4.4
通讯作者：
Ach, Thomas
中科院分区：
文献类型：
--
作者：
Bermond, Katharina;Wobbe, Christina;Ach, Thomas
目的.人视网膜色素上皮细胞（RPE）积累对自体荧光成像有意义的颗粒。细胞内积累和分布的知识是有限的。使用高分辨率显微镜技术，我们确定了每个细胞的颗粒总数，细胞内分布，以及与视网膜地形图和年龄相关的变化。在年轻（80岁，n = 7）供体中，使用结构照明显微镜（SIM）和共聚焦荧光显微镜对来自15个人RPE平坦载片的中央凹、中央凹周围和近周边的RPE细胞进行成像。使用定制的FIJI插件，用计算机辅助标记颗粒，基于形态学和自发荧光特性进行分类，并分析细胞内分布、每个细胞的总数和颗粒密度。共分析了450个RPE细胞体中的193，096个颗粒。基于自发荧光特性、大小和组成，RPE颗粒表现出九种不同的表型（脂褐质，两种;黑素脂褐质，五种;黑素体，两种），可通过SIM区分。总体而言，脂褐素（低在中央凹，但随着偏心率和年龄的增加）和黑素脂褐素（均匀分布在所有三个位置，没有年龄相关的变化）是主要的颗粒类型。黑素体的代表性不足，由于在平坦的安装顶突起的次优可视化。中心凹RPE细胞体内的低脂褐素和高黑素脂褐素含量以及中心凹周围的丰富脂褐素表明不同的起源，可能与覆盖的光感受器的群体（中心凹，仅视锥;中心凹周围，最高的视杆密度）相关。该系统分析提供了对RPE细胞和颗粒生理学的进一步了解，并将颗粒负荷与细胞自发荧光联系起来，为临床眼底自发荧光的解释提供了亚细胞基础。
PURPOSE. The human retinal pigment epithelium (RPE) accumulates granules significant for autofluorescence imaging. Knowledge of intracellular accumulation and distribution is limited. Using high-resolution microscopy techniques, we determined the total number of granules per cell, intracellular distribution, and changes related to retinal topography and age.METHODS. RPE cells from the fovea, perifovea, and near-periphery of 15 human RPE flat mounts were imaged using structured illumination microscopy (SIM) and confocal fluorescence microscopy in young (80 years, n = 7) donors. Using custom FIJI plugins, granules were marked with computer assistance, classified based on morphological and autofluorescence properties, and analyzed with regard to intracellular distribution, total number per cell, and granule density.RESULTS. A total of 193,096 granules in 450 RPE cell bodies were analyzed. Based on autofluorescence properties, size, and composition, the RPE granules exhibited nine different phenotypes (lipofuscin, two; melanolipofuscin, five; melanosomes, two), distinguishable by SIM. Overall, lipofuscin (low at the fovea but increases with eccentricity and age) and melanolipofuscin (equally distributed at all three locations with no age-related changes) were the major granule types. Melanosomes were under-represented due to suboptimal visualization of apical processes in flat mounts.CONCLUSIONS. Low lipofuscin and high melanolipofuscin content within foveal RPE cell bodies and abundant lipofuscin at the perifovea suggest a different genesis, plausibly related to the population of overlying photoreceptors (fovea, cones only; perifovea, highest rod density). This systematic analysis provides further insight into RPE cell and granule physiology and links granule load to cell autofluorescence, providing a subcellular basis for the interpretation of clinical fundus autofluorescence.