Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face
Andreas Pollreisz1, Jeffrey D Messinger2, Kenneth R Sloan3
1Ophthalmology, Medical University Vienna, Vienna, Austria.
Abstract:
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 ML; 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 μm 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.
Insights
Serial section block-face scanning electron microscopy (SBFSEM) quantified melanosomes, lipofuscin, and melanolipofuscin in retinal pigment epithelium (RPE). SBFSEM revealed hundreds of these granules per RPE cell, with spacing dependent on granule size.
Area of Science:
- Ophthalmology
- Cell Biology
- Microscopy
Background:
- The retinal pigment epithelium (RPE) plays a crucial role in photoreceptor maintenance and visual function.
- Understanding the ultrastructure of RPE cells, including the distribution of key organelles, is vital for comprehending visual processes and diseases.
Purpose of the Study:
- To evaluate the utility of serial section block-face scanning electron microscopy (SBFSEM) for detailed analysis of RPE cell ultrastructure.
- To quantify the number and three-dimensional distribution of melanosomes (M), lipofuscin (L), and melanolipofuscin (ML) within adult human RPE cell bodies.
Main Methods:
- SBFSEM was employed on epoxy-embedded macular tissue from four Caucasian donors.
- Automated ultramicrotomy and backscattered electron imaging generated aligned image stacks.
- Manual annotation identified cellular boundaries and the precise locations (x,y,z) of M, L, and ML granules.
Main Results:
- SBFSEM successfully visualized and quantified M, L, and ML within RPE cells.
- The number of granules varied significantly between donors, with lipofuscin being most abundant.
- Melanosomes were predominantly located near apical processes, and granule spacing was found to be regulated solely by granule size, with an effective radius of 1 μm for all granule types combined.
Conclusions:
- SBFSEM is an effective method for high-resolution ultrastructural analysis of RPE.
- Adult human RPE contains hundreds of melanosomes, lipofuscin, and melanolipofuscin.
- This quantitative data on granule distribution and spacing provides a foundation for future studies on RPE function, aging, and disease pathogenesis.
