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Andreas Pollreisz, Jeffrey D Messinger, Kenneth R Sloan, Emily Benson, Grahame J Kidd, Ursula Schmidt-Erfurth, Christine A Curcio; Visualizing retinal pigment epithelium (RPE) melanosomes, lipofuscin, melanolipofuscin (M, L, ML) in human eyes of different ages using 3-dimensional serial block face scanning electron microscopy (3D SBFSEM). Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the number and volumetric distribution of RPE M, L, and ML in normal eyes of young and aged adults, using 3D-SBFSEM of archived specimens originally prepared for transmission EM; elucidate mechanisms of organelle regulation; improve the interpretation of clinical RPE imaging.
Eyes of white donors (32F, 62F, 76F, 84M) with unremarkable macular morphology were preserved rapidly, post-fixed with osmium, and embedded in epoxy. In 3D SBFSEM, an epoxy block mounted in a Zeiss SigmaVP field emission SEM is sectioned using an in-chamber automated ultramicrotome that is part of the Gatan 3View system. After each serial 125 nm-thick section, electrons backscattered from the block face are captured, generating aligned and visualized tissue volumes. Granules and boundaries of individual RPE cells were apparent from image stacks, using alignment and orthogonal stack viewing tools in ImageJ (Figure).
From each donor, serial images of 300-700 sections containing 15-20 nuclei were examined. Many ML had fibrous interiors and electron-dense caps along the perimeter. L and ML were numerous in the basolateral 2/3 of cell bodies. Spindle-shaped M were present in the apical 1/3 of cell bodies and apical processes. Of interest was a diverse population of complex ML granules at all ages examined. The 32F sample was notable for abundant M in apical processes compared to older donors. Sloughed RPE in the 84M peripapillary area were spherical and contained smaller granules of the same types as cells in the RPE layer.
3D SBFEM is suitable for investigating the characteristic intracellular distribution of organelles in human RPE of different ages and morphologies. This information is important for testing hypotheses about organelle turnover and regulation in health, aging, and disease, and for understanding how RPE-specific signals are generated in various retinal imaging technologies including optical coherence tomography and autofluorescence.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Human RPE visualized by 3D SBFSEM. (A) Vertical cross-section of 84M with en face projection views of panels B and C indicated; (B) Junctional complexes (arrowheads) delimit individual cells; (C) Basolaterally located nuclei (arrowheads) are visible but individual cells not.
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