Autofluorescent Granules of the Human Retinal Pigment Epithelium: Phenotypes, Intracellular Distribution, and Age-Related Topography

Abstract

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 (≤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.

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.

Figure 1.

Granule subclassification. Nine different AF granule types were detectable in human RPE cells. For each type an exemplary SIM image and a schematic depiction are shown. For the SIM images, white pixels = high AF intensity and dark pixels = low AF intensity. Iso-AF was defined as the average AF intensity of the cytoplasm of the cell. Lipofuscin L1: round or oval granule exhibiting homogeneous AF; lipofuscin L2: accumulation of compacted inhomogeneous lipofuscin-like AF material larger than L1, with no further delineation of individual granules possible. Note that L2 (multiple lipofuscin-like material) is different from previously described granule aggregates in Ach et al. (accumulations of L1 granule type). Melanolipofuscin ML1: round, oval, or spindle-shaped granules with little to no AF in the center and full AF coating that varies in thickness (light blue - thin coating; dark blue -thick coating); melanolipofuscin ML2: similar to type 1 but with hypo- to iso-AF center; melanolipofuscin ML3: similar to type 1 and 2 but only partly coated with AF material; melanolipofuscin ML4: bull's-eye-shaped granule with AF center surrounded by hypo-AF material and an AF coating; melanolipofuscin ML5: large granule (>2 times L1) with hypo-AF core and weak to distinctive AF coating. Melanosome M1: spherical granule with an absence of both internal AF and AF coating; melanosome M2: spindle-shaped granule with an absence of both internal AF and AF coating. The color coding scheme shown here (L, yellow; ML1–3, blue; ML4, violet; ML5, dark blue; M, brown) is used for all subsequent figures. Scale bar: 1 µm.

Figure 2.

SIM and LSM imaging of RPE cells. At each location, identical RPE cells were imaged using both SIM (A) and LSM (B). Some lipofuscin granules were pushed toward the basolateral cell borders due to the cell nucleus; however, the relative hypoautofluorescent center of the RPE cells can be attributed to the high content of melanolipofuscin granules and does not exclusively represent the cell nucleus; also see Figure 2 in Starnes et al. The perifovea donor was a 51-year-old female.

Figure 3.

Absolute numbers of granules per RPE cell at the fovea, perifovea, and near-periphery for donor eyes ≤ 51 years and > 80 years. (A) RPE cells contain hundreds of granules in their cell bodies, with increasing number at greater age. Foveal RPE cells contained fewer L compared to cells from the other locations. Pure M were found only sporadically, likely due to inconsistent preservation of apical processes in whole mounts. A few large cells containing over 1000 granules were found at the perifovea and near-periphery. (B) ML subtypes ML4 and ML5 were low in number and spared the fovea. (C) For better illustration, M per cell are plotted with a different scale. The low number of melanosomes might be explained by the possibility of the absence of apical processes (due to preparation artifacts) or the fact that the characterization is based solely on the autofluorescence properties of the granules, meaning that melanosomes that show any kind of autofluorescence at the short wavelengths excitation are counted as melanolipofuscin.

Figure 4.

Granule distribution at the fovea, perifovea, and near-periphery. Percentages of L, M, ML1–3, ML4, and ML5 within the RPE cell bodies are plotted. Interestingly, the proportion of pure L was low at the fovea and increased at the perifovea and the near-periphery, peaking at the perifovea. The granule load of RPE cells at the fovea is mainly driven by ML, with ML types ML1–3 being the most abundant. Granules were characterized based on their short-wavelength autofluorescence properties.

Figure 5.

En face and cross-section views of the intracellular granule distribution. (A) The en face view shows the high L content in parafoveal and near-peripheral cells, whereas foveal cells contained only a few L. In some cells, L were located more toward cell borders, due to a centrally located nucleus (see also Fig. 2). For three male donors (36, 82, and 88 years old), the cell borders of the foveal cells were not distinguishable in the SIM images. Therefore, 10 square areas equal to the size of a typical foveal RPE cell were analyzed (see Methods). (B) ML1–3 was abundant throughout the cushion of organelles of autofluorescent relevance within the RPE cell body (Q1–Q3); however, L was located predominantly at the basolateral and basal parts of the RPE cell bodies (Q2, Q3). With increasing age, the deposition of L increases, especially at the perifovea and near-periphery. The fovea showed only few L granules and many ML granules. Only a few M were seen at the apical side of the RPE cell bodies (Q1). Within the basal parts of the RPE cell bodies (Q4), mitochondria are known to be abundant (not visible in the SIM autofluorescence imaging). Each analyzed granule was color coded and plotted (L, yellow; ML1–3, blue; ML4, violet; ML5, dark blue; M, brown dots). Because there were only a few M, ML4, and ML5, only a few violet or brown dots can be spotted. Red lines represent cell borders. Scale bar: 10 µm.

Figure 6.

Intracellular granule distribution. (A) SIM z-stacks of each cell were divided into three horizontally equally sized parts (upper, middle, and lower). The number of L, ML, M, ML4, and ML5 in each volume is shown. L were found mainly in the middle part, whereas ML, M, and ML4 were most abundant in the upper and middle part and ML5 in the middle and lower part. (B) Schematic depiction of an RPE cell (L, yellow; ML, blue; M, brown; nucleus, gray; mitochondria, violet). Note that the upper and lower scans begin and end with the first and last AF granules in and out of focus, which does not necessarily coincide with the apical and basal ends of the RPE cell.

Figure 7.

Relationship between cell area and number of granules per cell. Total number of granules per cell increased with larger cell areas for both age groups and at all locations. Foveal cells were smaller (175 ± 53 µm²) and contained fewer granules in total (322.1 ± 114.7). In general, cells at the perifovea and near -periphery occupied a larger area (perifovea, 225 ± 74 µm²; near-periphery, 218 ± 73 µm²), deposited more granules in total (perifovea, 508.9 ± 197.5; near-periphery, 456.3 ± 243.6), and showed a higher variability in granule load than foveal cells. The regression lines and 95% confidence intervals are shown for each location for both age groups.

Figure 8.

Proposed mechanism of ML and L development. M are present within RPE cells at birth. Unknown processes at the M surface might lead to marginal AF coatings or show focal hotspots of AF. Both scenarios lead to the ML phenotype (I), a combination of M and fluorophores of lipofuscin origin. Simultaneously, ML change their shape from spindle to round. Ongoing reactions form ML granules exhibiting a melanin core and progressive AF coating (II). This ratio (melanin/ fluorophores of lipofuscin origin) finally inverts, with AF material exceeding the enclosed melanin remnants (III). Further remodeling then leads to the formation of pure L granules. Our hypothesis is supported by the high heterogeneity of the ML1–3 group, the decreasing number of M and increasing number of L with age, and the mainly spindle-shaped granules (M and ML) apically and round granules (ML and L) basolaterally and basally (data not shown).