Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan,

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human retinal

pigment epithelium. IOVS 2014 Figure S1:

Fig. S1. Effect of fixation/preservation on A2E content of RPE/ choroid. The A2E content

of 4% paraformaldehyde (Pf)-fixed samples was elevated compared to fresh (Fr), untreated samples. Five mm diameter punches from peripheral (Per) tissues had a significantly higher A2E content compared to tissues from the macular area (M), as expected. 44 From this analysis we concluded that our assessment of total RPE AF, which includes A2E and other known and unknown bis-retinoids, was at least as good as fresh tissue and may even be superior because overall morphology is preserved. Others have reached similar conclusions about fixation and other tissue lipids. 86, 87

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pigment epithelium. IOVS 2014 Figure S2:

Fig. S2. Preserving foveal position during RPE-BrM flat mount preparation. The foveal

center (foveola) was preserved during RPE-BrM flat mount preparation, after the

neurosensory retina was removed. (A) The original tissue with the retina still on. The foveola (black arrow) is determined by the dark reddish spot (thinnest area of the overlying retina; reflected light from the choroid; trans-illumination image). 88 Ruby bead, 1 mm. (B) Removing

the retina and peeling-off choroid vessels and connective choroidal tissue results in a single-cell layer RPE-Bruch’s membrane mount (~ 20 µm thickness) (C). For imaging, the flat-mount was placed on a microscope slide and cover slipped (D).

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Fig. S2 (continued). Based on the original foveal position in (A) and additional landmarks

(e.g., tissue relaxation cuts), the foveal position was re-calculated by aligning the single images using a custom software tool (arrow in B–D). Tissue: 47-year-old female donor; red

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

retinal pigment epithelium. IOVS 2014 Figure S3:

Fig. S3. Voronoi tessellations for analysis of packing geometry and AF of individual RPE cells, sampled using an unbiased pattern. (A) A projection image of RPE

cytoskeleton (labeled with AlexaFluor 647) was post processed with a constrained iterative module with the advanced maximum likelihood estimation algorithm, 10 iterations (cellSens Dimension, Olympus) to highlight the labeled structures. For illustration, red was manually sharpened and brightened (Adobe Photoshop CS6). (B) A custom ImageJ plugin enabled each RPE cell center to be counted automatically (blue and red dots) with manual over-ride.

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Fig. S3. (continued). Three adjacent points form the vertices of a Delaunay triangle (blue

triangles). Voronoi polygons are formed from perpendicular lines, which bisect edges of a Delaunay triangle (green lines). All Voronoi regions (red borders, red cell centers) completely within a manually drawn lasso (black) were analyzed. (C) AF image from the same location as in (A). Excitation 488 nm, emission > 510 nm. Z-stack of multiple images; 0.4 µm step size. (D) The AF intensity (from C) of every pixel within a Voronoi region (from B) was summed and divided by the number of pixels within the region. The gray scale color code represents the mean AF per pixel [a.u., arbitrary units] within a Voronoi region. The AF intensity of a pixel is projected from a stack of intensities in the z-direction. (A-D): Tissue: 83-years-old female

donor, perifoveal region. (E) In this way, 75 to 90 areas on each flat mount were imaged using

the same sampling pattern, centered on the fovea (center of spiral pattern). At each eccentricity there are 8 equally spaced points. Eccentricities are closely spaced near the fovea and further apart in the periphery.

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Fig. S4. Individual quantified quantitative AF maps of all donors. AF is normalized to a

reference standard. The maps show high variability. In young donors, the AF signal is low in most cases. In older donors, there is still variability but overall AF increases. However, older age does not necessarily result in high AF intensity. The AF maps show a ring of higher AF intensity centered to the fovea for both age groups, more obvious in the > 80 years group. All maps were normalized to a fluorescence standard. Visualization conventions are the same as in Figure 1.

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pigment epithelium. IOVS 2014 Figure S5:

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Fig. S5. Individual numerical density maps of all donors. Numerical density appears

independent from age, as shown by maps of all tissues analyzed. A peak density can be found within the fovea in all flat mounts. No obvious loss of RPE cells occurs with age. Between maps, there is less variability. The highest cell density (≈ 10000 cells / mm2) was found in the fovea of an 82-year-old donor (central white area in the map). Each map results from 75 to 90 single RPE cytoskeleton images at predefined locations. Age of the donor is shown in the lower right corner of each panel. Visualization conventions are the same as in Figure 1. The different size and shapes of the maps result from variable dissection of the original tissues. The color bar shows the numerical density of RPE cells between 0 and 10000 cells/mm2 in increments of 625 cells/mm2.

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Fig. S6. The topography of RPE AF is related to the topography of overlying rod photoreceptors. Composite maps of AF (AF) intensity of human RPE in donors > 80 years

and previously published rod topography of human retina (modified from 19). Areas with highest AF are within 2 to 4 mm of the fovea, as are the highest numerical densities of rod photoreceptors. Since RPE AF originates in the photoreceptors, rods might have the highest impact on increasing RPE AF with age. Cones, which peak at the fovea (not shown in the rod plot), also contribute to RPE AF, because there is also AF detectable at the foveal rod-free zone. Visualization conventions for AF are the same as Figure 1. The color bar for rod topography shows increments of 12500 rods/mm2.

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pigment epithelium. IOVS 2014

Table S1. Donor characteristics. All donors were Caucasians.

Group Age Gender Death to Preservation

[years] time [hours]

≤ 51 16 male 2,4 29 male 2,3 36 male 4,1 40 female 2,8 40 female 3,4 41 female 1,8 47 female 2,3 49 male 0,8 50 female 1,5 51 female 3,4 > 80 82 male 1,4 83 female 2,1 83 female 4,2 83 female 1,2 83 female 1,5 85 female 2,5 85 female 0,9 86 male 2,9 88 male 3,6 90 female 1,3

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

retinal pigment epithelium. IOVS 2014.

Table S2. Flat mount sampling and cell analysis overview.

Group

RPE flat

mounts Areas imaged Cells analyzed

Total Fovea Perifovea Periphery Annulus 0 Annulus 1 Annulus 2

n n n n n n n n n

Total sample 20 1470 83330 16958 21942 44430 25425 6151 1865

≤ 51 group 10 735 40052 7444 9986 22622 11286 2649 1035

> 80 group 10 735 43278 9514 11956 21808 14139 3502 830

The table summarizes the number of RPE flat mounts and total number of locations where RPE was imaged. Locations were chosen in reference to the photoreceptor population in the overlying retina 19: fovea (0 to 600 µm from foveal center): cones only, perifovea (601 to 300 µm): highest rod:cone ratio, periphery (> 3001 µm): highest rod density. Locations were more closely spaced near the fovea where a gradient in RPE cell density was expected. 23, 29 Locations were analyzed in different retinal regions (fovea, perifovea, periphery, annulus 0 - 2). The annuli are centered on the fovea and have different radii, as specified for clinical quantitative AF. 45 The smaller number of sampling locations in the ≤51 group for fovea and perifovea results from RPE strongly adhering to the retina during dissection.

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

retinal pigment epithelium. IOVS 2014

Table S3. Studies reporting RPE cell density in humans and monkeys.

Author Year # of Eyes Donor Age Distribution Postmortem

Time

Exact Localization of

Foveola

Tissue Preparation RPE Cell Density Change with Age

Ts´o et Friedman 39

1968 10 4 months (gestation) to 96

years

not mentioned no bleached RPE/choroid flat

mount

de- and increase

Streeten 40 1969 90 4 months (gestation) to 74

years

not mentioned no in situ and whole mounts no change

Robb 89

1985 19 25 weeks (gestation) to 6

years

not mentioned no RPE flat mount no change

Dorey et al. 15 1989 19 2 weeks to 84 years < 12 hours no vertical histological sections

without split cell corrections

decrease Feeney-Burns 90

1990 35 91 to 101 years < 39 hours yes vertical histological sections

without split cell corrections

decrease

Gao et Hollyfield 23 1992 12 17 to 95 years < 3 hours yes en face histological sections no change

Watzke et al. 41 1993 20 13 years to 96 years not mentioned yes bleached RPE/choroid flat

mount

no change

Panda-Jonas et al. 38 1996 53 18 years to 85 years up to 29 hours no RPE punches decrease

Harman et al. 21 1997 21 12 to 89 years not mentioned no RPE/choroid flat mount temporal quadrant: increase;

other quadrants: no change DelPriore et al. 22

2002 22 19 to 84 years 14 to 24 hours no RPE/choroid flat mount overall: decrease ; within central

12.5 mm: no change

Snodderly et al. 29 2002 10 monkeys 7.6 to 33 years < 75 min yes RPE flat mount no change

Leung et al. 91 2004 15 monkeys 7 to 17 years 30 min to 1h yes vertical histological sections,

with split cell corrections

increase Morgan et al. 33

2009 3 humans, 2

monkeys

25 to 30 years (humans) n/a; in vivo

study

yes n/a; n/a

Current study 2014 20 16 to 51 (group ≤ 51) and 82

to 90 years (group > 80)

< 4.2 h yes RPE flat mount no overall net loss; increased

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

Table S4. Cell density, autofluorescence (AF) intensity, cell area, and number of neighbors at 3 canonical locations and 3 regional annuli. 45

Fovea Perifovea Periphery

≤ 51 Group > 80 Group ≤ 51 Group > 80 Group ≤ 51 Group > 80 Group

mean ± SD mean ± SD p value mean ± SD mean ± SD p value mean ± SD mean ± SD p value Cell density [cells/mm2 ] 6520 946 6405 1323 0,4616 5091 823 5304 1005 0,0433 4420 559 4478 622 0,1670 AF [a.u.] × 1000 min 9,48 5,54 13,93 9,67 0,0007 11,41 6,35 17,32 11,29 < 0,0001 10,91 6,43 17,00 10,94 < 0,0001 max 35,06 16,67 50,15 31,65 0,0005 38,23 17,63 51,64 28,95 < 0,0001 31,50 14,91 48,19 26,46 < 0,0001 mean 19,63 9,41 29,66 18,21 0,0001 23,64 11,21 34,19 19,31 < 0,0001 20,68 10,16 32,19 17,92 < 0,0001

Cell area [µm2] min 100,36 17,07 97,83 22,95 0,3585 125,70 23,39 113,14 20,89 < 0,0001 148,39 23,77 144,32 26,33 0,0271

max 244,18 92,10 279,83 84,44 0,0072 329,57 127,65 341,71 121,20 0,3699 369,66 86,53 387,94 103,82 0,0108

mean 157,33 29,16 162,43 33,26 0,2332 201,35 31,53 195,44 39,16 0,1364 230,04 30,72 227,95 34,22 0,3546

% % p value % % p value % % p value

Number of neighbors [%] ≤ 4 1,12 1,85 1,75 1,83 0,0189 1,35 1,62 2,11 2,01 0,0010 1,78 1,97 1,67 2,18 0,4164 5 20,09 4,58 23,86 4,34 < 0,0001 25,94 5,15 27,22 5,10 0,0300 27,53 5,43 27,32 5,03 0,5599 6 58,88 8,86 52,69 9,01 < 0,0001 49,63 8,74 46,07 7,66 0,0007 46,51 8,31 46,98 8,15 0,4099 7 18,03 4,19 18,03 4,61 0,9984 19,06 4,48 20,21 4,03 0,0199 20,30 5,11 19,69 4,89 0,0864 ≥ 8 1,88 1,98 3,67 2,43 < 0,0001 4,03 2,68 4,38 2,61 0,2169 3,87 2,70 4,34 2,92 0,0230

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Table S4 (continued)

Annulus 0 Annulus 1 Annulus 2

≤ 51 Group > 80 Group ≤ 51 Group > 80 Group ≤ 51 Group > 80 Group

mean ± SD mean ± SD p value mean ± SD mean ± SD p value mean ± SD mean ± SD p value Cell density [cells/mm2 ] 6174 1033 6209 1273 0,7802 4847 681 5300 844 0,0101 4473 592 4527 665 0,1911 AF [a.u.] × 1000 min 9,47 5,49 14,08 9,60 < 0,0001 11,90 5,95 17,38 10,67 0,0070 11,15 6,53 17,42 11,24 < 0,0001 max 36,04 16,97 50,24 30,53 < 0,0001 39,80 18,85 48,65 27,90 0,0859 32,23 15,29 49,16 27,11 < 0,0001 mean 20,25 9,66 30,06 17,95 < 0,0001 25,30 11,95 33,49 18,92 0,0221 21,16 10,39 32,98 18,43 < 0,0001

Cell area [µm2] min 105,96 20,22 100,54 22,27 0,0280 130,44 24,26 111,95 20,08 0,0008 146,27 24,19 141,03 26,82 0,0044

max 261,74 86,34 291,91 99,40 0,0073 360,59 211,90 333,31 79,06 0,3932 366,02 85,32 385,74 109,90 0,0050

mean 167,06 31,95 167,57 34,47 0,8871 210,47 30,56 192,81 27,24 0,0078 227,58 31,10 225,99 35,89 0,4631

% % p value % % p value % % p value

Number of neighbors [%] ≤ 4 1,06 1,69 1,80 2,03 0,0016 1,51 1,68 2,21 1,54 0,0449 1,76 1,94 1,74 2,15 0,8991 5 21,64 5,36 24,53 4,51 < 0,0001 25,95 4,62 27,77 4,25 0,0564 27,50 5,36 27,42 5,16 0,8083 6 56,62 9,75 51,24 8,75 < 0,0001 48,88 7,39 44,77 7,23 0,0129 46,64 8,24 46,71 8,12 0,8907 7 18,21 4,46 18,67 4,49 0,3468 19,42 3,61 20,38 4,00 0,2266 20,17 5,06 19,77 4,80 0,2122 ≥ 8 2,47 2,29 3,76 2,45 < 0,0001 4,24 2,84 4,86 2,65 0,2730 3,93 2,70 4,36 2,88 0,0253

Locations: fovea: 0 to 600 µm, perifovea: 601 to 3000 µm, periphery: ≥ 3001 µm from foveal center. Annulus: annulus 0: 0 to 1240 microns, annulus 1: 1241 to 2010 microns, annulus 2: 2011 to 2780 microns from foveal center. All data are based on mean values from each imaged sampling location.

Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

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Ach, Huisingh, McGwin, Messinger, Zhang, Bentley, Gutierrez, Ablonczy, Smith, Sloan, Curcio: Quantitative autofluorescence and cell density maps of the human

Table S5. Single cell analysis: Mean spacing, cell area, total autofluorescence (AF) per cell, and total AF per cell area at three locations and three annuli.

≤ 51 Group > 80 Group

n min max mean STD n min max mean STD

Mean spacing 7444 8,00 13,00 13,19 1,60 9514 6,27 29,08 13,41 2,08

Fovea Cell area 7444 57,17 888,54 150,78 37,92 9514 39,79 698,27 157,89 47,98

Total AF/cell 7444 7,74 1331,85 109,30 85,25 9514 4,82 1513,68 165,13 152,86

AF/Cell area 7444 0,07 2,93 0,69 0,42 9514 0,00 4,62 1,00 0,75

Mean spacing 9986 8,91 45,48 15,00 2,09 11956 8,11 36,75 14,66 2,30

Perifovea Cell area 9986 77,33 1719,59 196,68 54,16 11956 65,55 1106,02 188,48 59,09

Total AF/cell 9986 6,75 997,01 173,28 124,95 11956 7,08 1900,03 235,72 194,51

AF/Cell area 9986 0,06 3,02 0,85 0,48 11956 0,07 4,72 1,20 0,78

Mean spacing 22622 9,58 32,55 16,13 2,07 21808 7,98 36,26 16,07 2,26

Periphery Cell area 22622 81,81 876,73 225,38 55,48 21808 56,34 1011,23 223,89 61,45

Total AF/cell 22622 11,98 1230,20 169,32 115,45 21808 8,64 1996,62 265,32 187,38

AF/Cell area 22622 0,07 3,06 0,74 0,42 21808 0,03 4,42 1,19 0,75

Annulus0 Cell area 11286 57,17 888,54 160,88 43,01 14139 39,79 1106,02 162,81 50,50

Total AF/cell 11286 6,75 1331,85 121,29 95,43 14139 4,82 1513,68 174,48 156,96

AF/Cell area 11286 0,06 3,02 0,72 0,43 14139 0,04 4,62 1,02 0,74

Total AF/cell 2649 12,25 997,01 195,48 137,93 3502 7,08 1388,82 235,32 179,61

AF/Cell area 2649 0,09 3,02 0,93 0,52 3502 0,08 4,72 1,20 0,76

Total AF/cell 26117 11,42 1230,20 171,83 116,97 25637 8,64 1996,62 268,54 194,00