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Accepted Manuscript
Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment
epithelium using serial block face scanning electron microscopy
Andreas Pollreisz, Jeffrey D. Messinger, Kenneth R. Sloan, Tamara Mittermueller,
Alexandra S. Weinhandl, Emily K. Benson, Grahame J. Kidd, Ursula Schmidt-Erfurth,
Christine A. Curcio
PII:
S0014-4835(17)30614-0
DOI:
10.1016/j.exer.2017.10.018
Reference:
YEXER 7229
To appear in:
Experimental Eye Research
Received Date: 23 August 2017
Revised Date:
17 October 2017
Accepted Date: 17 October 2017
Please cite this article as: Pollreisz, A., Messinger, J.D., Sloan, K.R., Mittermueller, T., Weinhandl,
A.S., Benson, E.K., Kidd, G.J., Schmidt-Erfurth, U., Curcio, C.A., Visualizing melanosomes, lipofuscin,
and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron
microscopy, Experimental Eye Research (2017), doi: 10.1016/j.exer.2017.10.018.
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ACCEPTED MANUSCRIPT
For Experimental Eye Research
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Visualizing melanosomes, lipofuscin, and melanolipofuscin in human
retinal pigment epithelium using serial block face scanning electron
microscopy
Andreas Pollreisz 1; Jeffrey D. Messinger 2; Kenneth R. Sloan 2, 3; Tamara
Mittermueller 1; Alexandra S. Weinhandl 1; Emily K. Benson 4; Grahame J. Kidd 4, 5;
Ursula Schmidt-Erfurth 1; Christine A. Curcio 2
Ophthalmology, Medical University Vienna, Vienna, Austria; 2 Ophthalmology,
University of Alabama at Birmingham, Birmingham, AL, United States; 3 Computer
Science, University of Alabama at Birmingham, Birmingham, AL, United States; 4
Renovo Neural Inc., Cleveland, OH, United States; 5 Neurosciences, Cleveland Clinic,
Lerner Research Institute, Cleveland, OH, United States
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Running Head: Visualizing RPE organelles by SBFSEM
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Metrics: 4223 words in main text; 5 figures; 1 table; 1 supplementary figure
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Financial disclosure:
This work was supported by a Macula Society Research Grant, unrestricted funds to the
Department of Ophthalmology at University of Alabama at Birmingham from Research to
Prevent Blindness, Inc., and EyeSight Foundation of Alabama. Human tissues were
obtained with funds from NIH grant EY06109 (CAC), P30 EY003039, and International
Retinal Research Foundation
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Corresponding Address:
Christine A. Curcio, PhD; Department of Ophthalmology; EyeSight Foundation of
Alabama Vision Research Laboratories; 1670 University Boulevard Room 360;
University of Alabama School of Medicine; Birmingham AL 35294-0099; Ph
205.996.8682; F 205.934.3425; Email curcio@uab.edu
Conflict of Interest statement:
Emily Benson: Employment (Renovo Neural Inc.); Grahame J Kidd: Consultant (Renovo
Neural Inc.)
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Key words
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Retinal pigment epithelium, lipofuscin, melanosomes, melanolipofuscin, aging, human,
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autofluorescence, optical coherence tomography, electron microscopy, density recovery
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profile, packing geometry
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Abstract
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To assess serial section block-face scanning electron microscopy (SBFSEM) for retinal
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pigment epithelium (RPE) ultrastructure, we determined the number and distribution
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within RPE cell bodies of melanosomes (M), lipofuscin (L), and melanolipofuscin (ML).
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Eyes of 4 Caucasian donors (16M, 32F, 76F, 84M) with unremarkable maculas were
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sectioned and imaged using an SEM fitted with an in-chamber automated
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ultramicrotome. Aligned image stacks were generated by alternately imaging an epoxy
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resin block face using backscattered electrons, then removing a 125 nm-thick layer.
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Series of 249-499 sections containing 5-24 nuclei were examined per eye. Trained
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readers manually assigned boundaries of individual cells and x,y,z locations of M, L, and
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ML. A Density Recovery Profile was computed in three dimensions for M, L, and ML.
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The number of granules per RPE cell body in 16M, 32F, 76F, and 84M eyes,
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respectively, was 465 ± 127 (mean±SD), 305 ± 92, 79 ± 40, and 333 ± 134 for L; 13 ± 9;
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6 ± 7, 131 ± 55, and 184 ± 66 for ML; and 29 ± 19, 24 ± 12, 12 ± 7, and 7 ± 3 for M.
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Granule types were spatially organized, with M near apical processes. The effective
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radius, a sphere of decreased probability for granule occurrence, was 1 µm for L, ML,
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and M combined. In conclusion, SBFEM reveals that adult human RPE has hundreds of
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L, LF, and M and that granule spacing is regulated by granule size alone. When
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obtained for a larger sample, this information will enable hypothesis testing about
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organelle turnover and regulation in health, aging, and disease, and elucidate how RPE-
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specific signals are generated in clinical optical coherence tomography and
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autofluorescence imaging.
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Highlights
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• SBFSEM is a 3-D imaging technique for visualizing and numerating organelles in a
volume.
• We analyzed human retinal pigment epithelium from 4 donors of different ages.
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• RPE cell bodies had hundreds of lipofuscin, melanolipofuscin, and melanosomes.
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Introduction
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The retinal pigment epithelium (RPE) is a cuboidal epithelium of neuroectodermal origin
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located between the choroid and neurosensory retina, with demanding dual
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responsibilities in maintaining tissues on apical and basal aspects. (Booij et al., 2010;
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Shi et al., 2008). It is visible clinically by fundus autofluorescence and optical coherence
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tomography (OCT) due to prominent organelles of lysosomal origin - lipofuscin,
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melanolipofuscin and melanosomes (Bagshaw et al., 2005; Dell'Angelica et al., 2000;
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Orlow, 1995; Schraermeyer and Heimann, 1999), collectively referred to herein as
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“granules”.
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The literature on RPE organelle content in humans and non-human primates in situ that
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can inform imaging and theories of pathogenesis is surprisingly limited. Foundational
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classics from Feeney and Delori, some approaching 40 years old, described RPE
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lipofuscin ultrastructure and autofluorescence (Feeney, 1978) a topography resembling
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that of rod photoreceptors (Wing et al., 1978), and an increase of lipofuscin and re-
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positioning of melanosomes with age (Feeney-Burns et al., 1984; Weiter et al., 1986)
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(also seen by (Feher et al., 2006)). Studies in non-human primate established the
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dependence of normal RPE ultrastructure on diet (Feeney-Burns et al., 1981) and
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recently, provided an expanded range of organelle morphology (Gouras et al., 2011;
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Gouras et al., 2010). Single-section transmission EM (TEM) has been the visualization
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method of choice for definitive subcellular investigations such as these, for decades.
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(Feeney, 1974; Feeney-Burns et al., 1984) Recently, super-resolution structured
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illumination microscopy has demonstrated hundreds of autofluorescent organelles in
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individual human RPE cells viewed in a projection image (Ach et al., 2014). In contrast,
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only scores of RPE organelle are counted using single section TEM, due to limited
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sample volume. (Feeney-Burns et al., 1984; Feeney-Burns et al., 1981; Feher et al.,
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2006)
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Accurate assessments of the total number of RPE granules, proportions of each type,
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and three-dimensional packing geometry would help elucidate organelle regulation and
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potential for renewal and turnover, as well as provide a subcellular basis for
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understanding RPE maturation, aging, and diagnostic imaging characteristics. In this
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report, we evaluated the value of comprehensive three-dimensional (volume) electron
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microscopy images. The advantages of this method over two-dimensional imaging
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include not only the ability to analyze many more RPE granules but also to identify
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subclasses of heterogeneous granules based on three-dimensional shape, size, and
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distribution of electron-dense components. Aligning many serial thin sections is
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challenging due to between-section warping and paucity of registration landmarks. Serial
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block face scanning electron microscopy (SBFSEM) is a recently commercialized
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technology that robotically sections a resin block mounted in a field emission scanning
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EM. Aligned image stacks are generated by alternately imaging an epoxy resin block
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face using backscattered electrons, then removing a 125 nm-thick layer. The
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backscattered electrons are used to generate a stack of aligned images of a tissue
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volume, including large areas of outer retina. (Mills et al., 2015; Mustafi et al., 2011;
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Ohno et al., 2011; Tait et al., 2013) Alternate technologies for volume electron
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microscopy, e.g., the Automatic Tape Assisted Microtomy method (Hayworth et al.,
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2014), involve archiving cut sections and capturing images as an extra, separate step
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from tissue sectioning. Herein we report a validation of SBFSEM methods, initial counts,
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and packing geometry analysis of lipofuscin, melanolipofuscin, and melanosomes in
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RPE cell bodies in normal human foveas from four eyes from donors of different ages.
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Data will lay a groundwork for future studies of eyes with AMD previously characterized
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by light microscopy (Zanzottera et al., 2015a; Zanzottera et al., 2015b).
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Methods
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Tissue recovery, characterization, and preparation
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We used samples from human donor eyes with unremarkable maculas archived from a
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previous study (Curcio et al., 2001). Donor eyes were accessioned for research
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purposes from non-diabetic white donors to the Alabama Eye Bank during the period
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1996-2012. For this study, two eyes were chosen from younger adults (16M, 32F) and
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two eyes from older adults (76F, 84M), with death-to-preservation times of 3.53, 3.34,
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0.95, and 4.08 hours, respectively. Eyes were preserved by immersion in 1%
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paraformaldehyde (diluted from 40% stock made from paraformaldehyde) and 2.5%
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glutaraldehyde in 0.1M phosphate buffer following anterior segment excision. Eyes
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lacked chorioretinal pathology, as determined with ex vivo color fundus photography and
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internal globe examination (Curcio et al., 1998) and confirmed by histology (Curcio et al.,
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2001). To accentuate extracellular lipid in drusen as required by previous studies, all
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tissues were post-fixed with osmium tannic acid paraphenylenediamine (OTAP) (Curcio
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et al., 2001; Guyton and Klemp, 1988), then embedded in epoxy resin. (Curcio et al.,
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2011) Submicrometer-thick sections in the vertical plane were stained with toluidine
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blue. Silver-gold thin sections were used for diagnosis, orientation, and tissue quality
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assurance at UAB before tissue blocks were transferred to Renovo Neural (Cleveland
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OH) for SBFSEM processing in the same vertical plane. Samples were taken from the
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foveal rod-free zone.
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It should be noted that specific post-fixation and staining methods are recommended for
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SBFSEM, i.e., en bloc staining with high concentrations of heavy metal stains (Ellisman
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et al., 2011). We embarked on the studies reported herein using archival normal tissues
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prepared by a method originally chosen for visualization of extracellular lipid in AMD
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deposits, with the overall goal of eventually analyzing RPE granules in similarly prepared
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AMD eyes (Curcio et al., 2017). It should also be noted that there are abundant
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melanosomes within RPE apical processes (Krebs and Krebs, 1991; Steinberg et al.,
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1977) that cannot be enumerated when the retina is detached, as were 3 of our 4
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specimens.
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Serial block face scanning electron microscopy (SBFSEM)
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SBFSEM was performed using a Zeiss Sigma VP scanning electron microscope system
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equipped with a Gatan 3View in-chamber ultramicrotome and Gatan low-kilovolt
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backscattered electron detector. Samples were imaged at 1.5-2.5kV, and slices of 125
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nm thickness removed, over a 150 µm length of RPE and Bruch’s membrane (Figure 1).
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Images were collected in high vacuum mode, because charge mitigation benefits of
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lower vacuum operation with these sparsely stained samples was insufficient to justify
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longer acquisition times needed for adequate resolution. Stacks of 300-700 slices at 10
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nm/pixel resolution were collected robotically overnight, amounting to 30-100 GB of data.
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Individual images were smoothed, enhanced in contrast, downsampled by a factor of 4
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in x and y (i.e., original images of 8192x6144 pixels were reduced to 2096x3172), and
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assembled into a stack by a human observer. Any problematic slices (<5% of total) were
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removed manually and replaced by duplicates of adjacent slices to maintain proper
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spacing, and the stack was registered.
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Problematic slices were generally those with either insufficient or excessive contrast,
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due to debris re-deposition on the block face or detector, or image drift due to charging
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of the block face when mainly sparse fields are imaged. In our samples prepared for
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TEM, small local variations in staining or embedding resin can produce large changes in
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image quality when imaged by SEM.
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Data collection via Fiji/Image J plugins
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Our approach entailed manual detection and assignments made by trained human
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readers with software assistance for bookkeeping, visualization, and analysis, as distinct
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from automated detection and segmentation by an algorithm. Each specimen was
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surveyed to identify the largest group of clearly visible cells. Four image stacks (one per
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eye) containing nuclei of 5-24 completely sectioned cells were manipulated using
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alignment and orthogonal stack viewing techniques using two custom Java plug-ins for
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Fiji (Pietzsch et al., 2012). Readers manually delineated cell boundaries in an en face
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view at the level of the junctional complexes (plug-in “Pick_Cells”). Then they specified
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locations of classified lipofuscin, melanolipofuscin, and melanosome granules in three
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dimensions in a cross-sectional view using morphologic criteria explained in the Results
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(plug-in “Pick_Particles”). Selected particles were manually marked with dots at their 3D
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centers: x,y coordinates were determined by location in the central image, and z was
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derived from the location of that image in the stack. Both tasks were performed by a
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primary reader (TM) and a supervising editor (AP). During annotation, readers scrolled
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through the image stack in the z dimension. Previously marked granules were displayed
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to provide context and prevent duplicate assignments. An adjustable window in z
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allowed the reader to control the number of marked granules displayed. Each granule,
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typically visible in 8-9 cross-sections, was classified by type. The location and typing of
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granules could be proofread and edited. When readers were satisfied with the
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annotation, data were written to CSV files for processing by stand-alone custom Java
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programs. We performed inter-individual reproducibility tests to ensure accurate
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assessment of granule number and types by spot-checking three individual randomly
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chosen cells in each of the 4 samples by two different readers (authors TM and AW).
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Granule counts in each cell varied by ≤10% between readers.
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Data analysis via Fiji/Image J plugins
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Granules were specified in x,y,z space and assigned to a cell if the x,y location of the
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granules was inside the cell boundary. Cell boundaries, in turn, were defined by a single
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x,y polygon, typically defined at the level of the junctional complexes among individual
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RPE cells.
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Density (number of organelles per unit volume) and spacing (distance between
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organelles in µm) are reciprocal values. Density is important for understanding the
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concentration of fluorophores and/ or reflective organelle surfaces relevant to imaging.
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The regularity of granule spacing may be elucidative for understanding processes that
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distribute organelles in the cell.
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To demonstrate the utility of a three-dimensional dataset, the 2D Density Recovery
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Profile method (Rodieck, 1991) was extended to three dimensions to describe the
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distribution of RPE granules in a volumetric dataset. The original purpose of the 2D-DRP
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was to analyze “the spatial density of a set of points as a function of the distance of each
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of those points from all the others”, as applied to planar arrays of retinal neuronal cell
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bodies. In the original application, the outcome measure was an “effective radius” of a
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circular region of decreased probability of finding a neighboring neuron around a
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reference neuron. The 2D-DRP plotted density as a function of distance from each cell in
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the x,y plane. Distances were binned, with each bin corresponding to an annulus around
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a central point. In 3D-DRP the bins correspond to spherical shells, with distances from
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the reference point computed in three dimensions. To determine if organelles within RPE
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cells exhibited structured, regular packing, we extended the 2D-DRP to a 3D-DRP by
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replacing areas by volumes and replacing cells by granules. Thus, the “effective radius”
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describes a spherical region of decreased probability (“dead zone”) of finding a second
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granule around each granule. Additional details of the 3D-DRP analysis are given in the
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figure legend to Figure 5.
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To implement the 3D-DRP, a bounding box was constructed in each study eye to
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enclose all the reconstructed RPE cells and then trimmed on six sides to minimize edge
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effects. Each granule in the trimmed bounding box was considered in turn a “reference
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granule.” All granules located ≤4.0 µm from the reference granule were used to compute
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distances, which were then binned to create a frequency histogram. This was converted
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to a DRP by dividing the counts by the volumes of the spherical shells centered on the
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reference granule to produce densities. Average density was computed from the volume
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of the trimmed bounding box and the total count of granules inside that box.
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Results
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Results are based on a sample size of 2535 granules in 5 cells of a 16-year-old male,
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8023 granules in 24 cells of a 32-year-old female, 5541 granules in 25 cells of a 76-year-
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old female and 6278 granules in 12 cells of an 84-year-old male donor eye. Statistical
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treatment is limited to description only due to the small number of eyes.
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Figure 2 shows selected views from a volumetric dataset from the 84-year-old male eye,
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starting with a cross-sectional view of the RPE layer (Figure 2A). Boundaries of
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individual RPE cells are apparent from image stacks, using alignment and orthogonal
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stack viewing techniques via custom-built ImageJ plugins (Figure 2B-C). In en face
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projection views, individual cells are delimited by their borders, as outlined by the actin
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cytoskeleton (yellow arrowheads in Figure 2B; inset). Nuclei are spherical and basally
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located, representing a granule-free volume (Figure 2C; inset).
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Lipofuscin, melanosomes, and melanolipofuscin are visualizable in detail within the
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volumetric dataset (Figure 3) during assessment by readers. Lipofuscin granules
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(Figure 3A-D) in cross-sectional view are round or oval with homogenously electron-
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dense interiors. They may be present with or without an electron-dense rim or electron-
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dense condensations around the granule exterior resembling earmuffs and representing
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sites of presumed phosphatase activity (Feeney, 1978). Melanosomes (Figure 3E,F) are
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typically spindle-shaped with homogeneously high internal electron-density.
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Melanolipofuscin (Figure 3G,H) combines features of lipofuscin and melanosomes,
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containing an electron-dense spindle, sometimes ovoid and fibrillar, within a larger
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granule. Melanolipofuscin and lipofuscin could have superficially similar cross-sectional
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profiles in two-dimensional images, and these were disambiguated by viewing in three
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dimensions. For example, Figure 3A shows a circular and electron-dense
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melanolipofuscin cross-section that was differentiable from a melanosome (Figure 3E,F)
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by scrolling through multiple sections that clearly showed the different shapes (spherical
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vs spindle-shape) and cross-sectional areas (large vs small). Similarly, the electron-
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dense earmuff on a lipofuscin granule (Figure 3C), which appears as a blob in a
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glancing section, was differentiable from melanosomes (Figure 3E,F) and from other
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lipofuscin granules (Figure 3A-D).
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Overlaying results from manual assignment of granule types in cross-sectional images
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and delineating cell borders in en face views revealed the distribution of lipofuscin
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(green), melanolipofuscin (blue) and melanosomes (black) across RPE cells of each
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donor eye, displayed as projection images (Figure 3I-J). En face projection images of all
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the granules assigned in each study eye are available in Supplementary Figure 1. The
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number of lipofuscin, melanolipofuscin, and melanosomes per RPE cell body in each
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eye is presented in Table 1. The numerically predominant granule type in all four eyes
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was lipofuscin (mean ± standard deviation), with 465 ± 127 in the 16-year-old male, 305
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± 92 in the 32-year-old female, 79 ± 40 in the 76-year-old female and 333 ± 134 in the
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84-year-old male (Figure 3K-M, Table 1). Older eyes had more melanolipofuscin
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granules than the younger eyes (13 ± 9, 16-year-old male; 6 ± 7, 32-year-old female;
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131 ± 55, 76-year-old female; 184 ± 66, 84-year-old male). In contrast, older eyes had
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fewer melanosomes than younger eyes (29 ± 19, 16-year-old male; 24 ± 12, 32-year-old
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female; 12 ± 7, 76-year-old female; 7 ± 3 in the 84-year-old male).
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As RPE cell bodies are reconstructed in their entirety by SBSFEM, exact information
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regarding granule localization within the cell body can be obtained. The vertical
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disposition of different granules types within the layer of RPE cell bodies (apical-
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basolateral axis) is of particular interest. In Figure 4 each of the four study eyes are
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shown both as a single slice from the SBFSEM image stack and as a graphic. The
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graphic shows all graded particles with centers within 500 nm in depth of this index slice.
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Comparing the 32-year-old and 76-year-old female donor eyes in Figure 4 revealed that
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lipofuscin and melanolipofuscin granules, respectively, are numerous in the basolateral
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5/6 of cell bodies. Spindle-shaped melanosomes are spread more widely across the
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apical-to-basal extent of RPE cell bodies in the 76-year-old eye than they are in the
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other three eyes, where they localize to the apical one-third. For reference, cross-
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sectional projection images of all granules assigned throughout each study eye are
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available in Supplementary Figure 1.
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In Figure 5, separate DRP analyses for the three granule types are shown for each of
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the four study eyes. The DRP analysis computes average density in a local
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neighborhood rather than over a whole cell, and thus it can avoid other space-filling
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organelles such as nuclei and mitochondria. The effective radius for each type of
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granule, and for all the granules combined, is approximately 1 µm, in all four eyes.
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Secondary peaks in the DRP beyond the first peak, indicative of a medium range order
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in the spatial distribution of granules, were not observed.
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Discussion
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Our quantitative data based on novel three-dimensional volumetric datasets indicate
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hundreds of granules per individual RPE cell body in normal donor eyes, thus extending
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our observations using structured illumination microscopy. (Ach et al., 2015) In the
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current study, differentiation among lipofuscin, melanolipofuscin, and melanosomes was
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best achieved by visualizing each individual granule by three-dimensional
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reconstruction, as evident in training readers. For example, lipofuscin granules could be
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labeled as melanolipofuscin, because the SBFSEM slice did not pass through granule
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centers, and electron-dense condensations around granule exteriors were thus
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incorrectly interpreted as an electron-dense spindle in a granule core. Such false
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assignments become evident when scrolling through the volumetric dataset thoroughly.
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Similarly, a melanosome located perpendicular to the section plane could look like
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lipofuscin with homogenous electron-dense content. Visualizing granules in their entirety
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clarifies their true nature and leads to more comprehensive and ultimately, we believe,
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more accurate counts than two-dimensional visualization. A key requirement was
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excellent section alignment, because misalignments could lead to duplicate counts.
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Alignment, in turn, requires high-quality initial imaging by SBFSEM.
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In this initial investigation we observed the same organelles in OTAP-preserved human
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RPE as those described in osmium-preserved human (Biesemeier et al., 2011; Feeney,
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1978; Feeney-Burns et al., 1984; Ng et al., 2008; Warburton et al., 2007) and monkey
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RPE (Gouras et al., 2011; Gouras et al., 2008). Gouras and associates recently
333
illustrated in non-human primate two additional types of spherical organelles with
334
electron-lucent interior vacuoles and electron-dense surfaces, one of which lacked
335
melanin by elemental analysis, and we did not observe these, perhaps due to the
336
different post-fixation technique (Gouras et al., 2011; Gouras et al., 2008; Streeten,
337
1961). Lipofuscin is a diverse category that may well be subdivided into more granule
338
types, with further investigation using these methods. Specific questions of interest are
339
whether granules differ in cone-dominant fovea vs rod dominant extrafoveal regions
340
(Curcio et al., 1990) and in cells with strong vs weak spectroscopic signal for N-retinyl-N-
341
retinylidene-ethanolamine (A2E) (far periphery and macula, respectively) (Ablonczy et
342
al., 2013; Bhosale et al., 2009; Pallitto et al., 2015). Further, the Feeney-Burns model of
343
lipofuscin biogenesis suggests that lysosomes and melanosomes fuse to form
344
melanolipofuscin. With absolute numbers of each granule type in eyes of different ages,
345
it may be possible to test this model and others in human eyes and eyes of animal
346
models.
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Knowledge regarding RPE granule content dates to the early 1960s when irregularly-
349
shaped pigment granules were first described in EM images (Bairaiti and Orzalesi,
350
1963). Feeney and Streeten established the presence and ultrastructure of long-lasting
351
inclusion bodies, (Feeney et al., 1965; Streeten, 1961) and lipofuscin, melanin, and
352
melanolipofuscin granules were differentiated from each other (Feeney, 1978) (Feeney-
353
Burns et al., 1980). The mean number of specific organelles per RPE cell has been
354
previously reported in single sections only. Total melanosomes and lipofuscin were
355
reported as 23-34 and 14-20, respectively, for two normal macaque monkeys (Feeney-
356
Burns et al., 1984). In humans, the numbers of melanosomes, lipofuscin, and
357
melanolipofuscin profiles per RPE cell found in adults of three age groups ranged from
358
4.5-26.6, 11.1-29.9, and 0.9-14.1, respectively, with higher values in the macula and in
359
older eyes (Feeney-Burns et al., 1984). Also in humans, the number of lipofuscin
360
granules per RPE cell in normal eyes was reported as 10-22 and up to 30 in AMD eyes
361
of unspecified stage (Feher et al., 2006). Our granule counts in whole cell bodies cannot
362
be directly compared with these prior reports due to different outcome measures, i.e.,
363
the number of 3-dimensional particles per cell in our study vs the previously reported
364
number of 2-dimensional profiles per cell. Our counts are consistent, however, with the
365
in situ appearance of normal adult macular RPE cells, which are packed with granules
366
visualizable by high-resolution light microscopy (Ach et al., 2015). A three-dimensional
367
view of RPE cells provided by SBFSEM gives us not only a total number but also a more
368
detailed characterization of the granule type, because an individual granule could be
369
viewed in its entirety, typically through 8-9 125 nm-thick sections.
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The analysis of RPE granule distribution by a novel 3D DRP method suggests that a
372
strong factor governing the intracellular deployment of granules is the size of the
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granules themselves and not processes operative in the cytosol, at least in these normal
374
cells. Conversely the absence of secondary peaks in the 3D DRP suggests that granules
375
are randomly packed without patterns suggesting cytoskeletal engagement, such as
376
linear alignment along microtubules or intermediate filaments, not surprising given that
377
other organelles are present. Secondary peaks are characteristic of biological arrays
378
with a quasi-crystalline order and thus a modal spacing, as well documented for the
379
mosaic of cone photoreceptors (Cooper et al., 2016). We investigated this possibility
380
through 3D DRP analysis, because of the remarkably tight packing of autofluorescent
381
granules in healthy aged RPE (Figure 1 of (Ach et al., 2015)). Extension of the 2D DRP
382
to 3D demonstrates the type of analysis possible with 3D datasets made available by
383
SBFSEM. Future analyses with larger sample will include a detailed examination of the
384
intra-cellular spacing of organelles, both collectively and by granule type, with respect to
385
the plane of junctional complexes that divides apical from basolateral in this polarized
386
epithelium. This analysis will require a more precise delineation of the RPE cell
387
boundaries than was possible in this initial study. Further, regularity metrics that are
388
sensitive to dropout in a way that the DRP is not (Cooper et al., 2016) will be necessary
389
to quantify the concurrent aggregation and degranulation of autofluorescent lipofuscin
390
and melanolipofuscin typical of RPE cells in AMD eyes (Ach et al., 2015).
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Although our novel ultrastructural volumetric imaging techniques show many RPE
393
granules overall, it is premature on the basis of these data to reevaluate previously
394
reported major trends in aging, including a decrease in melanosomes and increase in
395
complex melanolipofuscin (Feeney, 1978; Feeney-Burns et al., 1984; Gouras et al.,
396
2011). In this initial sample we found that lipofuscin was the most common granule type,
397
overlapping total granule numbers for young and older adults, and an overall higher
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melanolipofuscin levels in the two older eyes (Feeney, 1978; Feeney-Burns et al., 1984;
399
Gouras et al., 2011). Although the greatest increase in lipofuscin reportedly occurs
400
before 30 years of age (Wing et al., 1978), it was nonetheless surprising that the 16-
401
year-old donor eye had so much lipofuscin. Further, assuming that later-appearing
402
melanolipofuscin develops from melanosomes, the latter might increase by as much as
403
10-fold over adulthood, a radical notion considering that RPE melanogenesis is widely
404
considered to terminate at birth. Indeed, prenatal RPE melanogenesis helps specify
405
retinal lamination and projections to the brain (Iwai-Takekoshi et al., 2016). The
406
possibility of a slow rate of postnatal RPE melanogenesis, reminiscent of brain
407
neuromelanin (Zecca et al., 2002), is not yet definitively excluded (Schraermeyer and
408
Heimann, 1999). Answering these questions will require a larger number of specimens,
409
and particularly specimens that also include apical processes, for a total accounting of
410
melanosomes.
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Quantifying RPE organelle number and packing characteristics are important for
413
validating the signal source for precise new diagnostic imaging technologies such as
414
quantitative (blue) autofluorescence (Delori et al., 2011; Gliem et al., 2016), near-
415
infrared autofluorescence for melanin (Keilhauer and Delori, 2006; Sparrow et al., 2015),
416
and OCT-based technologies especially polarization sensitive OCT (Götzinger et al.,
417
2008; Pircher et al., 2011; Sugita et al., 2013). The remnants of phagocytized
418
photoreceptor outer segments accumulate throughout life as lipofuscin and
419
melanolipofuscin granules, which contain fluorophores rich in bisretinoid derivatives of
420
vitamin A (Feeney-Burns et al., 1980; Katz et al., 1996; Sparrow and Boulton, 2005;
421
Weiter et al., 1986). Retinal OCT reflections are mainly generated by boundaries of
422
refractive index that strongly scatter light (Mie scattering) (Baumann et al., 2012; Wilson
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et al., 2007; Zhang et al., 2013), e.g. as induced by RPE-specific spindle-shaped
424
melanosomes, mitochondria and lysosomes. Imaging-histology correlation studies have
425
directly demonstrated that cells and cellular fragments containing these organelles are
426
reflective (Balaratnasingam et al., 2017; Chen et al., 2016; Pang et al., 2015; Zanzottera
427
et al., 2015b). Retinal tissues may contain structures that either alter or preserve the
428
polarization state of light detected by optical coherence tomography instruments.
429
Recently Baumann and coworkers compared the depolarization characteristics of the
430
RPE/choroid complex with corresponding histologic serial sections in rat eyes and found
431
a strong correlation between depolarization units - a way to quantify the polarization-
432
scrambling properties of tissue - and melanin pigmentation (Baumann et al., 2015). To
433
aid the interpretation of technologies that visualize RPE organelles, it is essential to
434
determine the absolute number of intracellular lipofuscin, melanolipofuscin, and
435
melanosomes in individual RPE cells in intact tissue (as opposed to isolated cells in
436
culture) (Burke et al., 1996; Sreekumar et al., 2016). This goal has been elusive, mainly
437
due to technical limitations of single section TEM, which SBFSEM can now address.
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Strengths of this initial project include the large dataset newly available from individual
440
RPE cells, short post-mortem tissues, and the three-dimensional visualization of
441
granules allowing for accurate manual assessment of granule number, type and location.
442
Limitations include the small number of eye specimens, retinal detachment that impacts
443
our ability to enumerate melanosomes in apical processes, reduced contrast in these
444
archival tissues, and difficulty in specifying each cell’s boundaries in three dimensions.
445
Nevertheless, we achieved our aim of introducing SBFSEM for imaging and quantifying
446
RPE ultrastructure in a volumetric dataset. Thus, we lay a firm foundation for future
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investigations of RPE organelles in numerous AMD eyes previously characterized at the
448
light microscopy level (Zanzottera et al., 2015a; Zanzottera et al., 2015b).
449
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Conflict of Interest
452
GJK and EKB are employees of Renovo Neural Inc, a contract research organization
453
that provides SBFSEM on a fee-for-service basis.
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Table 1: Lipofuscin, melanolipofuscin, and melanosomes per RPE cell
body, determined by serial block face scanning electron microscopy
Lipofuscin
Melanolipofuscin Melanosomes
Donor age,
N
Mean
SD
Mean
SD
Mean
SD
sex cells
16, M
5
465
127
13
9
29
19
32, F
24
305
92
6
7
24
12
76, F
25
79
40
131
55
12
7
84, M
12
333
134
184
66
7
3
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Figure Captions
458
Figure 1: Principles of serial section block-face scanning electron microscopy
459
(SBFSEM)
460
SBFSEM enables acquisition of ultrastructural information over a large retinal tissue
461
block. Epoxy-embedded samples (A) are sectioned and imaged using a SEM fitted with
462
an in-chamber automated ultramicrotome. Aligned image stacks were generated by
463
alternately imaging an epoxy resin block face using backscattered electrons (B), then
464
removing a 125 nm-thick layer (C).
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Figure 2: Human RPE in cross-sectional and en face projection views by SBFSEM
468
Retina was detached from the RPE post-mortem in this 84-year-old male donor. Wisps
469
of RPE apical processes are still apparent (upper surface of cells). A. Vertical cross-
470
section indicating levels of en face projection views (panels B and C). B. Individual cells
471
are delimited (inset) by actin cytoskeleton associated with junctional complexes, at the
472
apical side of the RPE cell. C. Basolaterally located nuclei (arrowheads), with details of
473
granules in the inset.
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Figure 3: Types, arrangement, and number of RPE granules in adult human eyes.
477
A-H. Three types of RPE granules identified. A-D. Lipofuscin granules show
478
homogeneous electron-dense filled circular or oval profile with or without an electron-
479
dense rim (A,D) or electron-dense condensations resembling earmuffs around the
480
granule perimeter (B,C). E,F. Melanosomes are spindle-shaped and electron-dense.
481
G,H. Melanolipofuscin combines features of lipofuscin and melanosomes, i.e., electron-
482
dense, spindle-shaped, and sometimes fibrillar cores, within larger granules, and with
483
(H) or without (G) electron-dense rims. I,J. Arrangement of granule types. In the 32-
484
year-old female donor (I), lipofuscin (green) is predominant, and in the 76-year-old
485
female donor (J), melanolipofuscin (blue) is predominant. For context, a projection
486
image of all the granules assigned for all the cells is available in Supplementary Figure
487
1. Scale bars, 10 µm. K,L,M. Distribution of granule types in RPE cells of eyes of
488
different ages. Shown are means (“+”) with 95% confidence intervals, per RPE cell.
489
Note that the graphs have different vertical scales.
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Figure 4: Apical-to-basal disposition of different granule types in study eyes.
492
Two panels are shown for each of the 4 study eyes (16M, 32F, 76F, 84M). The top panel
493
of each pair shows a single slice from the SBFSEM image stack. The bottom panel
494
shows particles with centers designated by manual grading within 500 nm (in the image
495
stack) of the single index slice. Because granules were classified and positions assigned
496
at the slice of best visibility for each individual granule, locations of circles in the graphic
497
representation may or may not correspond precisely with granules in the slice. For
498
context, a projection image of all the granules assigned for all the cells is available in
499
Supplementary Figure 1. Granules are color-coded as indicated. The scale bar for
500
sample 84M applies to all panels. Retina was attached to the RPE in sample 76F and
501
detached for the other eyes. Lipofuscin is predominant in all four eyes. The 16-year-old
502
male and the 32-year-old female have more apically placed melanosomes than the other
503
two. In the 76-year-old female, melanolipofuscin is the prevailing granule type, and
504
melanosomes are located more basally than in the younger eyes. The 84-year-old male
505
has high numbers of melanolipofuscin and lipofuscin.
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Figure 5: Three-Dimensional Density Recovery Profiles for RPE granules
509
Density Recovery Profiles for each granule type (lipofuscin - green, melanosomes -
510
black, and melanolipofuscin - blue) were computed separately and shown for each of the
511
four cases. The two-dimensional Density Recovery Profile method (Rodieck, 1991) was
512
extended to three dimensions. A bounding box was constructed to enclose all
513
reconstructed cells and trimmed on six sides to minimize edge effects. Each granule in
514
this box was considered, in turn, a “reference granule.” All granules located ≤4.0 µm
515
from the reference granule were used to compute distances, which were then binned to
516
create a frequency histogram. This was converted to a DRP by dividing counts by the
517
volumes of spherical shells centered on the reference granule to produce densities.
518
Average density, shown by the horizontal lines, was computed over the entire trimmed
519
bounding box. “Effective radius” (indicated by the vertical line) indicates a sphere of
520
decreased probability for granule occurrence close to the reference granule (“dead
521
space”). The effective radius is computed by considering the cumulative DRP and noting
522
the distance at which the cumulative density matches the global average density. The
523
3D DRP used here directly extends the 2D DRP introduced by Rodieck (Rodieck, 1991)
524
by replacing areas of concentric circular annuli with volumes of concentric spherical
525
shells. This effective radius is a measure of a “forbidden volume” surrounding each
526
granule, within which there is a reduced probability of finding a second granule. Our
527
results indicate that the size of these forbidden volumes is primarily due to the physical
528
size of the granule. Also, in quasi-crystalline structures, the DRP may exhibit secondary
529
modes, indicating distances to first neighbors, second neighbors, etc, which we do not
530
see in our 3D DRPs, indicating that granule arrangement is random, subject only to
531
physical constraints based on granule size. In all cases, the only difference in DRPs for
532
young and old eyes is the number of particles; the overall shapes are the same.
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Supplementary Figure 1:
535
Projection images showing all
536
lipofuscin (green),
537
melanolipofuscin (blue), and
538
melanosomes (black), as
539
assigned by graders, in the
540
cross-sectional and en face
541
views. Selected cross-
542
sectional views and en face
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images are also shown in
544
Figures 3 and 4.
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