Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy

To assess serial section block-face scanning electron microscopy (SBFSEM) for retinal pigment epithelium (RPE) ultrastructure, we determined the number and distribution within RPE cell bodies of melanosomes (M), lipofuscin (L), and melanolipofuscin (ML). Eyes of 4 Caucasian donors (16M, 32F, 76F, 84M) with unremarkable maculas were sectioned and imaged using an SEM fitted with an in-chamber automated ultramicrotome. Aligned image stacks were generated by alternately imaging an epoxy resin block face using backscattered electrons, then removing a 125 nm-thick layer. Series of 249-499 sections containing 5-24 nuclei were examined per eye. Trained readers manually assigned boundaries of individual cells and x,y,z locations of M, L, and ML. A Density Recovery Profile was computed in three dimensions for M, L, and ML. The number of granules per RPE cell body in 16M, 32F, 76F, and 84M eyes, respectively, was 465 127 (mean SD), 305 +/- 92, 79 +/- 40, and 333 +/- 134 for L; 13 +/- 9; 6 7, 131 +/- 55, and 184 +/- 66 for MLZ and 29 +/- 19, 24 +/- 12, 12 +/- 7, and 7 +/- 3 for M. Granule types were spatially organized, with M near apical processes. The effective radius, a sphere of decreased probability for granule occurrence, was 1 mu m for L, ML, and M combined. In conclusion, SBFEM reveals that adult human RPE has hundreds of L, LF, and M and that granule spacing is regulated by granule size alone. When obtained for a larger sample, this information will enable hypothesis testing about organelle turnover and regulation in health, aging, and disease, and elucidate how RPE-specific signals are generated in clinical optical coherence tomography and autofluorescence imaging.