Chemical composition of melanosomes, lipofuscin and melanolipofuscin granules of human RPE tissues

Energy-filtered analytical transmission electron microscopy was used to image the ultrastructure and determine quantitatively the chemical composition of pigment granules of the choroid and retinal pigment epithelium of two healthy human donors, aged 68 and 85 years. The electron microscopy preparation procedure did not affect the autofluorescence of melanolipofuscin and lipofuscin granules, since staining was omitted during sample preparation. Oval melanosomes, melanolipofuscin and lipofuscin granules were observed, having sizes of about 1.5 μm × 0.5 μm, and were analyzed using energy-dispersive X-ray microanalysis and electron energy loss spectroscopy. Up to now, these pigments could only be identified by scattering contrast in bright field images, with melanosomes having dark contrast and lipofuscin being much brighter. High-precision energy-dispersive X-ray microanalysis of pigment granules (>15,000 integrated counts in the oxygen K_α peak) yielded minimum detectable mole fractions of about 0.02 at% for copper and zinc. For the first time, quantitative analytical electron microscopy yielded the chemical composition of the different pigments without prior isolation from the tissue. This is important to better understand physical and chemical properties of the pigments and their metabolism and turnover.The composition of melanosomes and lipofuscin can clearly be distinguished by the applied methods. Melanosomes were the pigments with largest oxygen (about 5 at%) and nitrogen (about 10 at%) mole fractions. The S/N ratio determination demonstrated a high pheomelanin content of the melanosomes. Lipofuscin had a significantly smaller oxygen mole fraction (about 4 at%) and nitrogen was found to be only slightly above the limit of detection (0.4 at%). For comparison, the cytoplasm contained oxygen and nitrogen mole fractions of 3 at% and 0.8 at%. Bright field images showed melanolipofuscin granules having a core-shell structure with a dark inner and a bright outer fraction. The dark fraction had a chemical composition close to the melanosomes and the composition of the bright fraction could be distinguished from that of lipofuscin due to a significantly increased nitrogen mole fraction in the melanolipofuscin granule. For all pigments observed the oxygen mole fraction yielded a positive correlation with the calcium mole fraction as previously established for melanosomes. Only lipofuscin contained measurable phosphorus mole fractions, which also correlated positively with oxygen.In lipofuscin, mole fractions of nitrogen were significantly smaller than in melanosomes and only indicated a small fraction of proteins. In contrast, the phosphorus mole fraction was significantly larger indicating the presence of significant amounts of phospholipids.Copper and zinc mole fractions were larger than 0.1 at% in the melanosomes, but were below the detection limit in the lipofuscin granules. Compared to melanosomes of monkeys and rats analyzed beforehand, human retinal pigment epithelium melanosomes contained the highest amount of zinc, which even exceeded the calcium mole fraction. Trace elements like zinc are of great importance for metabolism and anti-oxidative mechanisms and also play a role in the progression of age related macular degeneration. They can now be investigated by quantitative analytical electron microscopy.     

Autofluorescent Organelles Within the Retinal Pigment Epithelium in Human Donor Eyes With and Without Age-Related Macular Degeneration

PurposeHuman retinal pigment epithelium (RPE) cells contain lipofuscin, melanolipofuscin, and melanosome organelles that impact clinical autofluorescence (AF) imaging. Here, we quantified the effect of age-related macular degeneration (AMD) on granule count and histologic AF of RPE cell bodies.MethodsSeven AMD-affected human RPE-Bruch''s membrane flatmounts (early and intermediate = 3, late dry = 1, and neovascular = 3) were imaged at fovea, perifovea, and near periphery using structured illumination and confocal AF microscopy (excitation 488 nm) and compared to RPE-flatmounts with unremarkable macula (n = 7, >80 years). Subsequently, granules were marked with computer assistance, and classified by their AF properties. The AF/cell was calculated from confocal images. The total number of granules and AF/cell was analyzed implementing a mixed effect analysis of covariance (ANCOVA).ResultsA total of 152 AMD-affected RPE cells were analyzed (fovea = 22, perifovea = 60, and near-periphery = 70). AMD-affected RPE cells showed increased variability in size and a significantly increased granule load independent of the retinal location (fovea: P = 0.02, perifovea: P = 0.04, and near periphery: P < 0.01). The lipofuscin fraction of total organelles decreased and the melanolipofuscin fraction increased in AMD, at all locations (especially the fovea). AF was significantly lower in AMD-affected cells (fovea: <0.01, perifovea: <0.01, and near periphery: 0.02).ConclusionsIn AMD RPE, lipofuscin was proportionately lowest in the fovea, a location also known to be affected by accumulation of soft drusen and preservation of cone-mediated visual acuity. Enlarged RPE cell bodies displayed increased net granule count but diminished total AF. Future studies should also assess the impact on AF imaging of RPE apical processes containing melanosomes.