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Invest Ophthalmol Vis Sci. 2020 May 11;61(5):35. doi: 10.1167/iovs.61.5.35.
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.
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 (≤51 years, n = 8) and older (>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.
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.
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.
人类视网膜色素上皮（RPE）细胞内含有大量对自发荧光成像有重要意义的颗粒。然而，目前我们对这些颗粒的细胞内积累和分布情况知之甚少。本研究采用高分辨率显微镜技术，旨在确定每个细胞中的颗粒总数、细胞内分布，并探讨其与视网膜拓扑结构和年龄的相关性。
我们对来自 15 例人 RPE 平铺标本的中心凹、中心凹旁和近周边区的 RPE 细胞进行了结构光照明显微镜（SIM）和共聚焦荧光显微镜检查。我们将年龄≤51 岁（n = 8）和>80 岁（n = 7）的年轻和老年供体的 RPE 细胞分别分为两组，并使用定制的 FIJI 插件，在计算机辅助下对颗粒进行标记，根据形态学和自发荧光特性对其进行分类，并分析其细胞内分布、每个细胞的总数和颗粒密度。
共分析了 450 个 RPE 细胞中的 193096 个颗粒。基于自发荧光特性、大小和组成，RPE 颗粒表现出 9 种不同的表型（脂褐素 2 种，黑素脂褐素 5 种，黑素体 2 种），可通过 SIM 进行区分。总体而言，脂褐素（中心凹处含量较低，但随离中心凹距离的增加和年龄的增长而增加）和黑素脂褐素（在三个部位的分布均匀，且与年龄无关）是主要的颗粒类型。由于平铺标本中顶突的可视化效果不理想，黑素体的数量相对较少。
中心凹 RPE 细胞内的脂褐素含量较低，黑素脂褐素含量较高，而周边区则富含脂褐素，这表明其起源不同，可能与上方感光细胞的数量有关（中心凹仅有锥细胞，周边区则有最高密度的杆状细胞）。本系统性分析进一步深入了解了 RPE 细胞和颗粒的生理学，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。
