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 Kα 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.

► Combined quantitative EELS and EDX yields the chemical composition of RPE pigments.
► Melanosomes and (melano-)lipofuscin granules can be distinguished by the methods.
► Lipofuscin lacks high N mole fractions, therefore protein content is low.
► The core of melanolipofuscin has the same elemental composition as melanosomes.
► Melanolipofuscin shell composition is lipofuscin-like except elevated N mole fraction.