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Andreas Pollreisz, Martina Neschi, Kenneth R Sloan, Ursula Schmidt-Erfurth, Christine A Curcio, Dennis M. Dacey; Visualizing melanosomes (M) in apical processes (AP) of human retinal pigment epithelium (RPE) using volumetric serial block-face scanning electron microscopy (SBFSEM). Invest. Ophthalmol. Vis. Sci. 2018;59(9):1506. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
RPE melanosomes are important signal sources for clinical imaging including optical coherence tomography, fundus autofluorescence, and color fundus photography. We sought to study for the first time the full complexity of M distribution and number within AP of human RPE using SBFSEM to obtain a 3-dimensional (3D) volume of cells reconstructed in their entirety.
A whole globe of a 28-year-old male Caucasian organ donor via rapid organ recovery. The enucleated eye was transected at the limbus, drained of vitreous, and placed in warm oxygenated culture medium (Ames). The retina was dissected from the sclera while maintained in this medium at 37°C before primary fixation. An epoxy-embedded sample of perifoveal RPE was sectioned and imaged using a field emission SEM fitted with an in-chamber automated ultramicrotome. An aligned image stack was generated by alternately imaging the resin block face using backscattered electrons, then removing an 80 nm-thick layer. The image stack was annotated by expert readers for cell body (CB) features, AP, and M using TrakEM (ImageJ) and a pen display (Wacom).
The 3D dataset showed exquisite preservation of cellular membranes and ultrastructure allowing the reconstruction of entire RPE cells including AP. M are typically spindle-shaped with homogeneously high internal electron-density. In 3 completely reconstructed RPE cells, AP containing M were tracked to their respective RPE CB. The number of M per RPE cell was 111 (a binucleate RPE cell), 47, and 26. The mean length and diameter of M was 2378±618 nm and 865±137 nm, respectively. The majority of M filled the entire cross section of its AP. Our initial count suggests that M in AP outnumbers those found in the CB as reported in our recent publication using similar technology. An ongoing study currently addresses the distribution of M between AP and CB. Surprisingly, AP in these 3 cells also contained some lipofuscin (n=26, 2, 9, respectively).
Our SBFSEM approach allowed the 3D reconstruction of entire human RPE and revealed that AP contain a sizeable number of M. When obtained for a larger sample size, these results will impact interpretation of clinical imaging of retina and pathophysiology of macular diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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