Atlas of Human Retinal Pigment Epithelium Organelles Significant for Clinical Imaging.

Purpose: To quantify organelles impacting imaging in the cell body and intact apical processes of human retinal pigment epithelium (RPE), including melanosomes, lipofuscin-melanolipofuscin (LM), mitochondria, and nuclei. Methods: A normal perifovea of a 21-year-old white male was preserved after rapid organ recovery. An aligned image stack was generated using serial block-face scanning electron microscopy and was annotated by expert readers (TrakEM, ImageJ). Acquired measures included cell body and nuclear volume (n = 17); organelle count in apical processes (n = 17) and cell bodies (n = 8); distance of cell body organelles along a normalized apical-basal axis (n = 8); and dimensions of organelle-bounding boxes in apical processes in selected subsamples of cell bodies and apical processes. Results: In 2661 sections through 17 cells, apical processes contained 65 ± 24 melanosomes in mononucleate (n = 15) and 131 ± 28 in binucleate cells (n = 2). Cell bodies contained 681 ± 153 LM and 734 ± 170 mitochondria. LM was excluded from the basal quartile, and mitochondria from the apical quartile. Lengths of melanosomes, LM, and mitochondria, respectively were 2305 ± 528, 1320 ± 574, and 1195 ± 294 nm. The ratio of cell body to nucleus volume was 4.6 ± 0.4. LM and mitochondria covered 75% and 63%, respectively, of the retinal imaging plane. Conclusions: Among RPE signal sources for optical coherence tomography, LM and mitochondria are the most numerous reflective cell body organelles. These and our published data show that most melanosomes are in apical processes. Overlapping LM and previously mitochondria cushions may support multiple reflective bands in cell bodies. This atlas of subcellular reflectivity sources can inform development of advanced optical coherence tomography technologies.

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 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.

A quantitative approach to identify morphological features relevant for visual function in ranibizumab therapy of neovascular AMD.

PURPOSE: To quantitatively analyze morphological features in eyes with neovascular AMD (nAMD) at baseline, after 12 months, and after 24 months of intravitreal ranibizumab treatment and to perform a structure/function correlation. METHODS: Eyes with treatment-naïve nAMD were treated with intravitreal ranibizumab according to a standardized dosing regimen over 2 years and followed continuously in a prospective study design. The central foveal area of 1000 µm (horizontal)×960 µm (vertical) of spectral-domain optical coherence tomography (SD-OCT) volume scans was evaluated quantitatively (using proprietary software) for the following pathologies: alteration of the external limiting membrane (ELM), alteration of the ellipsoid zone, subretinal fluid, pigment epithelium detachment, drusen, intraretinal cysts, subretinal mass, and subretinal pigment epithelium mass. The total area of each pathology was calculated in mm2 at baseline and after 1 and 2 years of ranibizumab therapy and correlated with BCVA results. RESULTS: In total, 480 central SD-OCT scans of 20 consecutive patients were evaluated. In the multivariate regression analysis, the area of ELM alteration, the area of intraretinal cysts, and foveal retinal thickness were significant variables influencing visual acuity at baseline (R=-0.827; R2=0.684; P<0.001). The area of ELM alteration was the only significant factor to be directly associated with visual acuity at 12 months (R=-0.846; R2=0.716; P<0.001) and 24 months (R=-0.778; R2=0.606; P<0.001). CONCLUSIONS: The integrity of the ELM appears to be the most important feature correlating with visual acuity in native nAMD as well as nAMD treated with intravitreal ranibizumab at each time interval, but not prospectively. In general, no significant predictors for an individual gain or loss in mid- (12 months) or long-term BCVA results (24 months) were found by OCT.