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Purpose: For structure-function research at the transition of aging to age-related macular degeneration, we refined the current consensus optical coherence tomography (OCT) nomenclature and evaluated a novel review software for investigational high-resolution OCT imaging (HR-OCT; <3 µm axial resolution)., Method: Volume electron microscopy, immunolocalizations, histology, and investigational devices informed a refined OCT nomenclature for a custom ImageJ-based review tool to assess retinal band visibility. We examined effects on retinal band visibility of automated real-time averaging (ART) 9 and 100 (11 eyes of 10 healthy young adults), aging (10 young vs 22 healthy aged), and age-related macular degeneration (AMD; 22 healthy aged, 17 early (e)AMD, 15 intermediate (i)AMD). Intrareader reliability was assessed., Results: Bands not included in consensus nomenclature are now visible using HR-OCT: inner plexiform layer (IPL) 1-5, outer plexiform layer (OPL) 1-2, outer segment interdigitation zone 1-2 (OSIZ, including hyporeflective outer segments), and retinal pigment epithelium (RPE) 1-5. Cohen's kappa was 0.54-0.88 for inner and 0.67-0.83 for outer retinal bands in a subset of 10 eyes. IPL-3-5 and OPL-2 visibility benefitted from increased ART. OSIZ-2 and RPE-1,2,3,5 visibility was worse in aged eyes than in young eyes. OSIZ-1-2, RPE-1, and RPE-5 visibility decreased in eAMD and iAMD compared to healthy aged eyes., Conclusions: We reliably identified 28 retinal bands using a novel review tool for HR-OCT. Image averaging improved inner retinal band visibility. Aging and AMD development impacted outer retinal band visibility., Translational Significance: Detailed knowledge of anatomic structures visible on OCT will enhance precision in research, including AI training and structure-function analyses.

Purpose: In AMD, rod-mediated dark adaptation (RMDA) at 5° eccentricity is slower in eyes with subretinal drusenoid deposits (SDDs) than in eyes without. Here we quantified SDD burden using supervised deep learning for comparison to vision and photoreceptor topography., Methods: In persons ≥60 years from the Alabama Study on Early Age-Related Macular Degeneration 2, normal, early AMD, and intermediate AMD eyes were classified by the AREDS nine-step system. A convolutional neural network was trained on 55°-wide near-infrared reflectance images for SDD segmentation. Trained graders annotated ground truth (SDD yes/no). Predicted and true datasets agreed (Dice coefficient, 0.92). Inference was manually proofread using optical coherence tomography. The mean SDD area (mm2) was compared among diagnostic groups (linear regression) and to vision (age-adjusted Spearman correlations). Fundus autofluorescence images were used to mask large vessels in SDD maps., Results: In 428 eyes of 428 persons (normal, 218; early AMD, 120; intermediate AMD, 90), the mean SDD area differed by AMD severity (P < 0.0001): 0.16 ± 0.87 (normal), 2.48 ± 11.23 (early AMD), 11.97 ± 13.33 (intermediate AMD). Greater SDD area was associated with worse RMDA (r = 0.27; P < 0.0001), mesopic (r = -0.13; P = 0.02) and scotopic sensitivity (r = -0.17; P < 0.001). SDD topography peaked at 5° superior, extended beyond the Early Treatment of Diabetic Retinopathy Study grid and optic nerve, then decreased., Conclusions: SDD area is associated with degraded rod-mediated vision. RMDA 5° (superior retina) probes where SDD is maximal, closer to the foveal center than the rod peak at 3 to 6 mm (10.4°-20.8°) superior and the further eccentric peak of rod:cone ratio. Topographic data imply that factors in addition to rod density influence SDD formation.

Background: Drusen and drusenoid deposits are a hallmark of age-related macular degeneration (AMD). Nowadays, a multimodal retinal imaging approach enables the detection of these deposits. However, quantitative data on subretinal drusenoid deposits (SDDs) are still missing. Here, we compare the capability of en-face drusen and SDD area detection in eyes with non-exudative AMD using conventional imaging modalities versus Retro mode imaging. We also quantitatively assess the topographic distribution of drusen and SDDs. Methods: In total, 120 eyes of 90 subjects (mean age ± standard deviation = 74.6 ± 8.6 years) were included. Coherent en-face drusen and SDD areas were measured via near-infrared reflectance, green (G-) and blue (B-) fundus autofluorescence (AF), and Retro mode imaging. Drusen phenotypes were classified by correlating en-face drusen areas using structural high-resolution spectral domain optical coherence tomography. The topographic distribution of drusen was analyzed according to a modified ETDRS (Early Treatment of Diabetic Retinopathy Study) grid. Intraclass correlation coefficient (ICC) analysis was applied to determine the inter-reader agreement in the SDD en-face area assessment. Results: The largest coherent en-face drusen area was found using Retro mode imaging with a mean area of 105.2 ± 45.9 mm 2 (deviated left mode (DL)) and 105.4 ± 45.5 mm 2 (deviated right mode (DR)). The smallest en-face drusen areas were determined by GAF (50.9 ± 42.6 mm 2 ) and BAF imaging (49.1 ± 42.9 mm 2 ) ( p < 0.001). The inter-reader agreement for SDD en-face areas ranged from 0.93 (DR) to 0.70 (BAF). The topographic analysis revealed the highest number of SDDs in the superior peripheral retina, whereas sub-retinal pigment epithelium drusen were mostly found in the perifoveal retina. Retro mode imaging further enabled the detection of the earliest SDD stages. Conclusions: Retro mode imaging allows for a detailed detection of drusen phenotypes. While hundreds/thousands of SDDs can be present in one eye, the impact of SDD number or volume on AMD progression still needs to be evaluated. However, this new imaging modality can add important knowledge on drusen development and the pathophysiology of AMD.

Purpose: In aging and early-intermediate age-related macular degeneration (AMD), rod-mediated dark adaptation (RMDA) slows more at 5° superior than at 12°. Using optical coherence tomography angiography (OCTA), we asked whether choriocapillaris flow deficits are related to distance from the fovea., Methods: Persons ≥60 years stratified for AMD via the Age-Related Eye Disease Study's nine-step system underwent RMDA testing. Two adjacent 4.4° × 4.4° choriocapillaris OCTA slabs were centered on the fovea and 12° superior. Flow signal deficits (FD%) in concentric arcs (outer radii in mm, 0.5, 1.5, 2.2, 4.0, and 5.0 superior) were correlated with rod intercept time (RIT) and best-corrected visual acuity (BCVA)., Results: In 366 eyes (170 normal, 111 early AMD, 85 intermediate AMD), FD% was significantly worse with greater AMD severity in all regions (overall P < 0.05) and poorest under the fovea (P < 0.0001). In pairwise comparisons, FD% worsened with greater AMD severity (P < 0.05) at distances <2.2 mm. At greater distances, eyes with intermediate, but not early AMD differed from normal eyes. Foveal FD% was more strongly associated with longer RIT at 5° (r = 0.52) than RIT at 12° (r = 0.39) and BCVA (r = 0.21; all P < 0.0001). Choroidal thickness was weakly associated with longer RIT at 5° and 12° (r = 0.10-0.20, P < 0.05) and not associated with AMD severity., Conclusions: Reduced transport across the choriocapillaris-Bruch's membrane-retinal pigment epithelium complex, which contributes to drusen formation under the macula lutea (and fovea), may also reduce retinoid resupply to rods encircling the high-risk area. FD% has potential as a functionally validated imaging biomarker for AMD emergence.

The fovea of the human retina, a specialization for acute and color vision, features a high concentration of cone photoreceptors. A pit on the inner retinal aspect is created by the centrifugal migration of post-receptoral neurons. Foveal cells are specified early in fetal life, but the fovea reaches its final configuration postnatally. Pre-term birth retards migration resulting in a small pit, a small avascular zone, and nearly continuous inner retinal layers. To explore the involvement of Müller glia, we used serial-section electron microscopic reconstructions to examine the morphology and neural contacts of Müller glia contacting a single foveal cone in a 28-year-old male organ donor born at 28 weeks of gestation. A small non-descript foveal avascular zone contained massed glial processes that included a novel class of 'inner' Müller glia. Similar to classic 'outer' Müller glia that span the retina, inner Müller glia have bodies in the inner nuclear layer (INL). These cells are densely packed with intermediate filaments and insert processes between neurons. Unlike 'outer' Müller glia, 'inner' Müller glia do not reach the external limiting membrane but instead terminate at the outer plexiform layer. One completely reconstructed inner cell ensheathed cone pedicles and a cone-driven circuit of midget bipolar and ganglion cells. Inner Müller glia outnumber foveal cones by 1.8-fold in the outer nuclear layer (221,448 vs. 123,026 cells/mm 2 ). Cell bodies of inner Müller glia outnumber those of outer Müller glia by 1.7-fold in the INL (41,872 vs. 24,631 cells/ mm 2 ). Müller glia account for 95 and 80% of the volume of the foveal floor and Henle fiber layer, respectively. Determining whether inner cells are anomalies solely resulting from retarded lateral migration of inner retinal neurons in pre-term birth requires further research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kar, Singireddy, Kim, Packer, Schalek, Cao, Sloan, Pollreisz, Dacey and Curcio.)

Purpose: Research on Alzheimer's disease (AD) and precursor states demonstrates a thinner retinal nerve fiber layer (NFL) compared to age-similar controls. Because AD and age-related macular degeneration (AMD) both impact older adults and share risk factors, we asked if retinal layer thicknesses, including NFL, are associated with cognition in AMD., Methods: Adults ≥ 70 years with normal retinal aging, early AMD, or intermediate AMD per Age-Related Eye Disease Study (AREDS) nine-step grading of color fundus photography were enrolled in a cross-sectional study. Optical coherence tomography (OCT) volumes underwent 11-line segmentation and adjustments by a trained operator. Evaluated thicknesses reflect the vertical organization of retinal neurons and two vascular watersheds: NFL, ganglion cell layer-inner plexiform layer complex (GCL-IPL), inner retina, outer retina (including retinal pigment epithelium-Bruch's membrane), and total retina. Thicknesses were area weighted to achieve mean thickness across the 6-mm-diameter Early Treatment of Diabetic Retinopathy Study (ETDRS) grid. Cognitive status was assessed by the National Institutes of Health Toolbox cognitive battery for fluid and crystallized cognition. Correlations estimated associations between cognition and thicknesses, adjusting for age., Results: Based on 63 subjects (21 per group), thinning of the outer retina was significantly correlated with lower cognition scores (P < 0.05). No other retinal thickness variables were associated with cognition., Conclusions: Only the outer retina (photoreceptors, supporting glia, retinal pigment epithelium, Bruch's membrane) is associated with cognition in aging to intermediate AMD; NFL was not associated with cognition, contrary to AD-associated condition reports. Early and intermediate AMD constitute a retinal disease whose earliest, primary impact is in the outer retina. Our findings hint at a unique impact on the brain from the outer retina in persons with AMD.

Introduction: The aims of the study were to describe baseline quantitative (short-wavelength) autofluorescence (qAF) findings in a large pseudophakic cohort at age-related macular degeneration (AMD)'s beginnings and to assess qAF8 as an outcome measure and evaluate Age-Related Eye Disease Study (AREDS) and Beckman grading systems., Methods: In the ALSTAR2 baseline cohort (NCT04112667), 346 pseudophakic eyes of 188 persons (74.0 ± 5.5 years) were classified as normal (N = 160 by AREDS, 158 by Beckman), early AMD (eAMD) (N = 104, 66), and intermediate AMD (iAMD) (N = 82, 122). Groups were compared via mean qAF intensities in a 6°-8° annulus (qAF8) and maps of differences between observations and the overall mean, divided by standard deviation (Z-score)., Results: qAF8 did not differ significantly among diagnostic groups by either stratification (p = 0.0869 AREDS; p = 0.0569 by Beckman). Notably, 45 eyes considered eAMD by AREDS became iAMD by Beckman. For AREDS-stratified eyes, Z-score maps showed higher centrally located qAF for normal, near the mean in eAMD, and lower values for iAMD. Maps deviated from this pattern for Beckman-stratified eyes., Conclusions: In a large sample of pseudophakic eyes, qAF8 does not differ overall from normal aging to iAMD but also does not capture the earliest AMD activity in the macula lutea. AREDS classification gives results more consistent with a slow decline in histologic autofluorescence than Beckman classification., (© 2024 S. Karger AG, Basel.)

Purpose: To longitudinally assess the impact of high-risk structural biomarkers for natural disease progression in non-exudative age-related macular degeneration (AMD) on spatially resolved mesopic and scotopic fundus-controlled perimetry testing., Methods: Multimodal retinal imaging data and fundus-controlled perimetry stimuli points were semiautomatically registered according to landmark correspondences at each annual visit over a period of up to 4 years. The presence of sub-RPE drusen, subretinal drusenoid deposits, pigment epithelium detachments (PEDs), hyper-reflective foci (HRF), vitelliform lesions, refractile deposits, and incomplete RPE and outer retinal atrophy (iRORA) and complete RPE and outer retinal atrophy (cRORA) were graded at each stimulus position and visit. Localized retinal layer thicknesses were extracted. Mixed-effect models were used for structure-function correlation., Results: Fifty-four eyes of 49 patients with non-exudative AMD (mean age, 70.7 ± 9.1 years) and 27 eyes of 27 healthy controls (mean age, 63.4 ± 8.9 years) were included. During study course, presence of PED had the highest functional impact with a mean estimated loss of -1.30 dB (P < 0.001) for mesopic and -1.23 dB (P < 0.001) for scotopic testing, followed by HRF with -0.89 dB (mesopic, P = 0.001) and -0.87 dB (scotopic, P = 0.005). Subretinal drusenoid deposits were associated with a stronger visual impairment (mesopic, -0.38 dB; P = 0.128; scotopic, -0.37 dB; P = 0.172) compared with sub-RPE drusen (-0.22 dB, P = 0.0004; -0.18 dB, P = 0.006). With development of c-RORA, scotopic retinal sensitivity further significantly decreased (-2.15 dB; P = 0.02). Thickening of the RPE-drusen-complex and thinning of the outer nuclear layer negatively impacted spatially resolved retinal sensitivity., Conclusions: The presence of PED and HRF had the greatest prognostic impact on progressive point-wise sensitivity losses. Higher predominant rod than cone-mediated localized retinal sensitivity losses with early signs of retinal atrophy development indicate photoreceptor preservation as a potential therapeutic target for future interventional AMD trials.

A progression sequence for age-related macular degeneration onset may be determinable with consensus neuroanatomical nomenclature augmented by drusen biology and eye-tracked clinical imaging. This narrative review proposes to supplement the Early Treatment of Diabetic Retinopathy Study (sETDRS) grid with a ring to capture high rod densities. Published photoreceptor and retinal pigment epithelium (RPE) densities in flat mounted aged-normal donor eyes were recomputed for sETDRS rings including near-periphery rich in rods and cumulatively for circular fovea-centered regions. Literature was reviewed for tissue-level studies of aging outer retina, population-level epidemiology studies regionally assessing risk, vision studies regionally assessing rod-mediated dark adaptation (RMDA), and impact of atrophy on photopic visual acuity. The 3 mm-diameter xanthophyll-rich macula lutea is rod-dominant and loses rods in aging whereas cone and RPE numbers are relatively stable. Across layers, the largest aging effects are accumulation of lipids prominent in drusen, loss of choriocapillary coverage of Bruch's membrane, and loss of rods. Epidemiology shows maximal risk for drusen-related progression in the central subfield with only one third of this risk level in the inner ring. RMDA studies report greatest slowing at the perimeter of this high-risk area. Vision declines precipitously when the cone-rich central subfield is invaded by geographic atrophy. Lifelong sustenance of foveal cone vision within the macula lutea leads to vulnerability in late adulthood that especially impacts rods at its perimeter. Adherence to an sETDRS grid and outer retinal cell populations within it will help dissect mechanisms, prioritize research, and assist in selecting patients for emerging treatments.

Purpose: Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles., Methods: A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods., Results: In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin., Conclusions: Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Purpose: Photoreceptor (PR) outer segments, retinal pigment epithelium apical processes, and inter-PR matrix contribute to the interdigitation zone (IZ) of optical coherence tomography (OCT). We hypothesize that this interface degrades over adulthood, in concert with a delay of rod mediated dark adaptation (RMDA). To explore this idea, we determined IZ discernibility and RMDA in younger and older adults., Methods: For this cross-sectional study, eyes of 20 young (20-30 years) and 40 older (≥60 years) participants with normal maculas according to the AREDS 9-step grading system underwent OCT imaging and RMDA testing at 5° superior to the fovea. Custom FIJI plugins enabled analysis for IZ discernibility at 9 eccentricities in 0.5 mm steps on one single horizontal B-scan through the fovea. Locations with discernible IZ met two criteria: visibility on B-scans and a distinct peak on a longitudinal reflectivity profile. The frequency of sites meeting both criteria was compared between both age groups and correlated with rod intercept time (RIT)., Results: The median number of locations with discernible IZ was significantly higher (foveal, 4 vs. 0, p  = 0.0099; extra-foveal 6 vs. 0, p  < 0.001) in eyes of young (26 ± 3 years) compared to older (73 ± 5 years) participants. For the combined young and older sample, the higher frequency of discernible IZ was correlated with shorter RIT (faster dark adaptation) ( r s  = -0.56, p  < 0.0001). This association was significant within young eyes ( r s  = -0.54; p  = 0.0134) and not within older eyes ( r s  = -0.29, p  = 0.706)., Conclusions: Results suggest that the interface between outer segments and apical processes degrades in normal aging, potentially contributing to delayed rod-mediated dark adaptation. More research is needed to verify an age-related association between IZ discernibility and rod-mediated dark adaptation. If confirmed in a large sample, IZ discernibility might prove to be a valuable biomarker and predictor for visual function in aging.

This study aimed to determine the retest variability of quantitative fundus autofluorescence (QAF) in patients with and without age-related macular degeneration (AMD) and evaluate the predictive value of patient reliability indices on retest reliability. A total of 132 eyes from 68 patients were examined, including healthy individuals and those with various stages of AMD. Duplicate QAF imaging was conducted at baseline and 2 weeks later across six study sites. Intraclass correlation (ICC) analysis was used to evaluate the consistency of imaging, and mean opinion scores (MOS) of image quality were generated by two researchers. The contribution of MOS and other factors to retest variation was assessed using mixed-effect linear models. Additionally, a Random Forest Regressor was trained to evaluate the extent to which manual image grading of image quality could be replaced by automated assessment (inferred MOS). The results showed that ICC values were high for all QAF images, with slightly lower values in AMD-affected eyes. The average inter-day ICC was found to be 0.77 for QAF segments within the QAF8 ring and 0.74 for peripheral segments. Image quality was predicted with a mean absolute error of 0.27 on a 5-point scale, and of all evaluated reliability indices, MOS/inferred MOS proved most important. The findings suggest that QAF allows for reliable testing of autofluorescence levels at the posterior pole in patients with AMD in a multicenter, multioperator setting. Patient reliability indices could serve as eligibility criteria for clinical trials, helping identify patients with adequate retest reliability., (© 2023. Springer Nature Limited.)

Purpose: Systemic chloroquine/hydroxychloroquine (CQ/HCQ) can cause severe ocular side effects including bull's eye maculopathy (BEM). Recently, we reported higher quantitative autofluorescence (QAF) levels in patients with CQ/HCQ intake. Here, QAF in patients taking CQ/HCQ in a 1-year follow-up is reported., Methods: Fifty-eight patients currently or previously treated with CQ/HCQ (cumulative doses 94-2435 g) and 32 age- and sex-matched healthy subjects underwent multimodal retinal imaging (infrared, red free, fundus autofluorescence [FAF], QAF [488 nm], and spectral-domain optical coherence tomography (SD-OCT). For analysis, custom written FIJI plugins were used for image processing, multimodal image stacks assembling, and QAF calculation., Results: Thirty patients (28 without BEM and 2 with BEM, age range = 25-69 years) were followed up (370 ± 63 days). QAF values in patients taking CQ/HCQ showed a significant increase between baseline and follow-up examination: 282.0 ± 67.9 to 297.7 ± 70.0 (QAF a.u.), P = 0.002. An increase up to 10% was observed in the superior macular hemisphere. Eight individuals (including 1 patient with BEM) had a pronounced QAF increase of up to 25%. Compared to healthy controls, QAF levels in patients taking CQ/HCQ were significantly increased (P = 0.04)., Conclusions: Our study confirms our previous finding of increased QAF in patients taking CQ/HCQ with a further significant QAF increase from baseline to follow-up. Whether pronounced QAF increase might predispose for rapid progression toward structural changes and BEM development is currently investigated in ongoing studies., Translational Relevance: In addition to standard screening tools during systemic CQ/HCQ treatment, QAF imaging might be useful in CQ/HCQ monitoring and could serve as a screening tool in the future.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear. This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids. AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions. This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Purpose: The purpose of this study was to analyze spatially resolved structural changes at retinal locations in presence (+) or absence (-) of hyper-reflective foci (HRF) in eyes with subretinal pigment epithelium (RPE) drusen in intermediate age-related macular degeneration (iAMD)., Methods: Patients with IAMD (n = 40; mean age = 69.7 ± 9.2 [SD] years) and healthy controls (n = 27; 64.2 ± 9.0) underwent spectral-domain optical-coherence-tomography imaging and fundus-controlled perimetry testing. After reviewing retinal layer segmentation, presence of HRF was annotated and retinal layer thicknesses (RLTs) extracted using ImageJ. Localized RLTs were compared between +HRF and -HRF positions. Univariate mixed linear models were used to investigate associations among RLT, HRF presence, and HRF size., Results: In iAMD eyes, a mean of 11.1 ± 12.5 HRF were detected with a peak abundance at 0.5 to 1.5 mm eccentricity to the fovea. At +HRF positions, outer nuclear layer (ONL; P = 0.0013, average difference = -12.4 µm) and retinal pigment epithelium drusen complex (RPEDC; P < 0.0001, +45.6 µm) thicknesses differed significantly compared to -HRF positions, even after correcting for accompanying drusen-related RPEDC layer thickening (P = 0.01). Mixed linear models revealed a significant association between increasing HRF area and decreasing ONL (association score = -0.17, P < 0.0001; 95% confidence interval [CI] = -0.22 to -0.11), and inner photoreceptor segments (IS) layer thicknesses (-0.08, P = 0.005; 95% CI = -0.14 to -0.03). Spearman rank correlation analysis yielded a significant correlation between total HRF area and mesopic (P = 0.015), but not scotopic (P = 0.305) retinal sensitivity losses., Conclusions: Descriptive analysis of this study demonstrated a predominant distribution of HRF at a foveal eccentricity of 0.5 to 1.5 mm, whereas further refined topographic analysis revealed a significant ONL layer thinning in presence of HRF even after correction for sub-RPE drusen presence compared to lesions in absence of HRF. Longitudinal studies are further needed to analyze the prognostic impact as well as the role of HRF presence in the context of iAMD.

Purpose: Quantification of retinal xanthophyll carotenoids in eyes with and without age-related macular degeneration (AMD) via macular pigment optical volume (MPOV), a metric for xanthophyll abundance from dual wavelength autofluorescence, plus correlations to plasma levels, could clarify the role of lutein (L) and zeaxanthin (Z) in health, AMD progression, and supplementation strategies., Design: Cross-sectional observational study (NCT04112667)., Participants: Adults ≥ 60 years from a comprehensive ophthalmology clinic, with healthy maculas or maculas meeting fundus criteria for early or intermediate AMD., Methods: Macular health and supplement use was assessed by the Age-related Eye Disease Study (AREDS) 9-step scale and self-report, respectively. Macular pigment optical volume was measured from dual wavelength autofluorescence emissions (Spectralis, Heidelberg Engineering). Non-fasting blood draws were assayed for L and Z using high-performance liquid chromatography. Associations among plasma xanthophylls and MPOV were assessed adjusting for age., Main Outcome Measures: Age-related macular degeneration presence and severity, MPOV in fovea-centered regions of radius 2.0° and 9.0°; plasma L and Z (μM/ml)., Results: Of 809 eyes from 434 persons (89% aged 60-79, 61% female), 53.3% eyes were normal, 28.2% early AMD, and 18.5% intermediate AMD. Macular pigment optical volume 2° and 9° were similar in phakic and pseudophakic eyes, which were combined for analysis. Macular pigment optical volume 2° and 9° and plasma L and Z were higher in early AMD than normal and higher still in intermediate AMD ( P  < 0.0001). For all participants, higher plasma L was correlated with higher MPOV 2° (Spearman correlation coefficient [R s ] = 0.49; P < 0.0001). These correlations were significant ( P  < 0.0001) but lower in normal (R s  = 0.37) than early and intermediate AMD (R s  = 0.52 and 0.51, respectively). Results were similar for MPOV 9°. Plasma Z, MPOV 2°, and MPOV 9° followed this same pattern of associations. Associations were not affected by supplement use or smoking status., Conclusions: A moderate positive correlation of MPOV with plasma L and Z comports with regulated xanthophyll bioavailability and a hypothesized role for xanthophyll transfer in soft drusen biology. An assumption that xanthophylls are low in AMD retina underlies supplementation strategies to reduce progression risk, which our data do not support. Whether higher xanthophyll levels in AMD are due to supplement use cannot be determined in this study., (© 2022 Published by Elsevier Inc. on behalf of American Academy of Ophthalmology.)

Purpose: The purpose of this study was to investigate quantitative autofluorescence (qAF8) in patients with and without early or intermediate age-related macular degeneration (AMD); to determine the impact of the aged crystalline lens and posterior capsular opacification (PCO)., Methods: In phakic and pseudophakic eyes ≥60 years, AMD status was determined by the Beckman system. PCO presence and severity was extracted from clinical records. qAF8 was calculated using custom FIJI plugins. Differences in qAF8, stratified by lens status, PCO severity, and AMD status, were analyzed using generalized estimating equations., Results: In 210 eyes of 115 individuals (mean age = 75.7 ± 6.6 years), qAF8 was lower in intermediate AMD compared to early AMD (P = 0.05). qAF8 did not differ between phakic and pseudophakic eyes (P = 0.8909). In phakic (n = 83) and pseudophakic (n = 127) eyes considered separately, qAF8 did not differ by AMD status (P = 0.0936 and 0.3494, respectively). Qualitative review of qAF images in phakic eyes illustrated high variability. In pseudophakic eyes, qAF8 did not differ with PCO present versus absent (54.5% vs. 45.5%). Review of implanted intraocular lenses (IOLs) revealed that 43.9% were blue-filter IOLs., Conclusions: qAF8 was not associated with AMD status, up to intermediate AMD, considering only pseudophakic eyes to avoid noisy images in phakic eyes. In pseudophakic eyes, qAF8 was not affected by PCO. Because blue-filter IOLs may reduce levels of exciting light for qAF8, future studies investigating qAF in eyes with different IOL types are needed., Translational Relevance: To reduce variability in observational studies and clinical trials requiring qAF8, pseudophakic participants without blue-filter IOLs or advanced PCO should be preferentially enrolled.

Purpose: Phenotype alterations of the retinal pigment epithelium (RPE) are a main characteristic of age-related macular degeneration (AMD). Individual RPE cell shape descriptors may help to delineate healthy from AMD-affected cells in early disease stages., Methods: Twenty-two human RPE flatmounts (7 eyes with AMD [early, 3; geographic atrophy, 1; neovascular, 3); 15 unaffected eyes [8 aged ≤51 years; 7 aged >80 years)] were imaged at the fovea, perifovea, and near periphery (predefined sample locations) using a laser-scanning confocal fluorescence microscope. RPE cell boundaries were manually marked with computer assistance. For each cell, 11 shape descriptors were calculated and correlated with donor age, cell autofluorescence (AF) intensity, and retinal location. Statistical analysis was performed using an ensemble classifier based on logistic regression., Results: In AMD, RPE was altered at all locations (most pronounced at the fovea), with area, solidity, and form factor being the most discriminatory descriptors. In the unaffected macula, aging had no significant effect on cell shape factors; however, with increasing distance to the fovea, area, solidity, and convexity increased while form factor decreased. Reduced AF in AMD was significantly associated with decreased roundness and solidity., Conclusions: AMD results in an altered RPE with enlarged and deformed cells that could precede clinically visible lesions and thus serve as early biomarkers for AMD onset. Our data may also help guide the interpretation of RPE morphology in in vivo studies utilizing high-resolution single-cell imaging., Translational Relevance: Our histologic RPE cell shape data have the ability to identify robust biomarkers for the early detection of AMD-affected cells, which also could serve as a basis for automated segmentation of RPE sheets.

Purpose: To evaluate hypotheses about the role of acquired vitelliform lesion (AVL) in age-related macular degeneration pathophysiology., Design: Laboratory histology study; retrospective, observational case series., Methods: Two donor eyes in a research archive with AVL and age-related macular degeneration were analyzed with light and electron microscopy for AVL content at locations matched to ex vivo B-scans. A retrospective, observational clinical cohort study of 42 eyes of 30 patients at 2 referral clinics determined the frequency of optical coherence tomography features stratified by AVL fate., Results: Histologic and clinical cases showed subretinal drusenoid deposit and drusen. Ultrastructural AVL components in 2 donor eyes included retinal pigment epithelium (RPE) organelles (3%-22% of volume), outer segments (2%-10%), lipid droplets (0.2%-12%), and a flocculent material (57%-59%). Of 48 AVLs (mean follow-up 46 ± 39 months), 50% collapsed to complete RPE and outer retinal atrophy, 38% were stable, 10% resorbed, and 2% developed neovascularization. The Early Treatment Diabetic Retinopathy Study grid central subfield contained 77% of AVLs. Hyperreflective foci, ellipsoid zone disruption, and hyperreflective thickening of the RPE-basal lamina-Bruch membrane band were common at maximum AVL expansion. Collapsing and noncollapsing AVLs had different growth rates (rapid vs slow, respectively)., Conclusions: AVL deposits contain unexpectedly low levels of RPE organelles and outer segments. Subfoveal predilection, reflectivity on optical coherence tomography, hyperautofluorescence, yellow color, and growth-regression phases suggest dysregulation of lipid transfer pathways specific to cone photoreceptors and supporting cells in formation of AVL deposit, analogous to drusen and subretinal drusenoid deposit. Prediction of AVL outcomes via growth rates should be confirmed in larger clinical studies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: To estimate macular pigment values in macular telangiectasia (MacTel) Type 2 in comparison with healthy subjects in the South Indian population across different spatial profiles and to quantify the regional differences of macular pigment optical density (MPOD) in MacTel Type 2., Methods: In this prospective cross-sectional study, healthy controls and patients diagnosed with various stages of MacTel Type 2 underwent MPOD measurement using dual-wavelength autofluorescence technique with Spectralis HRA + OCT., Results: Sixty eyes of 31 healthy subjects and 41 eyes of 22 MacTel type 2 patients were included. We found an overall decrease in MPOD values in MacTel type 2 patients (-0.109, -0.11, -0.001) in comparison with healthy subjects (0.38, 0.23, 0.06) at 1°, 2° & 6° foveal eccentricities (P < 0.001). In various stages of MacTel type 2, the mean MPOD was found to be higher in the peripheral region compared to the central region. We found a significantly lower mean MPOD in the central region in association with specific optical coherence tomography (OCT) parameters like inner retinal cavities (P = 0.035) and ellipsoid zone disruption (P = 0.034)., Conclusions: In MacTel type 2, MPOD distribution varies in different spatial profiles with higher MPOD levels in the peripheral region compared to the central region. The macular pigment levels are associated with inner retinal cavities and ellipsoid zone disruption seen on OCT., (© 2022. The Author(s).)

Purpose: Subretinal drusenoid deposits (SDD) first appear in the rod-rich perifovea and can extend to the cone-rich fovea. To refine the spatial relationship of visual dysfunction with SDD burden, we determined the topography of mesopic and scotopic light sensitivity in participants with non-neovascular AMD with and without SDD., Methods: Thirty-three subjects were classified into three groups: normal (n = 9), AMD-Drusen (with drusen and without SDD; n = 12), and AMD-SDD (predominantly SDD; n = 12). Mesopic and scotopic microperimetry were performed using 68 targets within the Early Treatment Diabetic Retinopathy Study grid, including points at 1.7° from the foveal center (rod:cone ratio, 0.35). Age-adjusted linear regression was used to compare mesopic and scotopic light sensitivities across groups., Results: Across the entire Early Treatment Diabetic Retinopathy Study grid and within individual subfields, the three groups differed significantly for mesopic and scotopic light sensitivities (all P < 0.05). The AMD-SDD group exhibited significantly decreased mesopic and scotopic sensitivity versus both the normal and the AMD-Drusen groups (all P < 0.05), while AMD-Drusen and normal eyes did not significantly differ (all P > 0.05). The lowest relative sensitivities were recorded for scotopic light levels, especially in the central subfield, in the AMD-SDD group., Conclusions: SDD-associated decrements in rod-mediated vision can be detected close to the foveola, and these deficits are proportionately worse than functional loss in the rod-rich perifovea. This finding suggests that factors other than the previously hypothesized direct cytotoxicity to photoreceptors and local transport barrier limitations may negatively impact vision. Larger prospective studies are required to confirm these observations.

Purpose: Human retinal pigment epithelium (RPE) cells contain lipofuscin, melanolipofuscin, and melanosome organelles that impact clinical autofluorescence (AF) imaging. Here, we quantified the effect of age-related macular degeneration (AMD) on granule count and histologic AF of RPE cell bodies., Methods: Seven AMD-affected human RPE-Bruch's membrane flatmounts (early and intermediate = 3, late dry = 1, and neovascular = 3) were imaged at fovea, perifovea, and near periphery using structured illumination and confocal AF microscopy (excitation 488 nm) and compared to RPE-flatmounts with unremarkable macula (n = 7, >80 years). Subsequently, granules were marked with computer assistance, and classified by their AF properties. The AF/cell was calculated from confocal images. The total number of granules and AF/cell was analyzed implementing a mixed effect analysis of covariance (ANCOVA)., Results: A total of 152 AMD-affected RPE cells were analyzed (fovea = 22, perifovea = 60, and near-periphery = 70). AMD-affected RPE cells showed increased variability in size and a significantly increased granule load independent of the retinal location (fovea: P = 0.02, perifovea: P = 0.04, and near periphery: P < 0.01). The lipofuscin fraction of total organelles decreased and the melanolipofuscin fraction increased in AMD, at all locations (especially the fovea). AF was significantly lower in AMD-affected cells (fovea: <0.01, perifovea: <0.01, and near periphery: 0.02)., Conclusions: In AMD RPE, lipofuscin was proportionately lowest in the fovea, a location also known to be affected by accumulation of soft drusen and preservation of cone-mediated visual acuity. Enlarged RPE cell bodies displayed increased net granule count but diminished total AF. Future studies should also assess the impact on AF imaging of RPE apical processes containing melanosomes.

Purpose: To investigate differences in quantitative autofluorescence (qAF) imaging measurements between eyes with and without large drusen, and whether qAF measurements change over time in the eyes with large drusen., Methods: Eighty-five eyes from participants with bilateral large drusen and 51 eyes from healthy participants underwent qAF imaging at least once, and the age-related macular degeneration participants were reviewed 6-monthly. Normalized grey values at 9° to 11° eccentricity from the fovea were averaged to provide a summary measure of qAF values (termed qAF8)., Results: In a multivariable model, qAF8 measurements were not significantly different between age-related macular degeneration eyes with large drusen and healthy eyes (P = 0.130), and qAF8 measurements showed a decline over time in the age-related macular degeneration eyes (P = 0.013)., Conclusion: These findings add to the body of evidence that qAF levels are not increased in eyes with large drusen compared with healthy eyes, and qAF levels show a significant decline over time in the age-related macular degeneration eyes. These findings highlight how the relationship between qAF levels and retinal pigment epithelium health does not seem to be straightforward. Further investigation is required to better understand this relationship, especially if qAF levels are to be used as an outcome measure in intervention trials.

Background: Cells of the retinal pigment epithelium (RPE) accumulate different kinds of granules (lipofuscin, melanolipofuscin, melanosomes) within their cell bodies, with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation. High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration (AMD); however, this has not been confirmed in histology so far. Here, based on our previous dataset of RPE granule characteristics, we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence (AF) intensities., Methods: RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy (HR-SIM) and laser scanning microscopy (LSM). Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules. In addition, total AF intensity per cell and granule density (number of granules per cell area) were determined. For the final analysis, RPE cells with total granule number below 5 th or above the 95 th percentile, or a total AF intensity ± 1.5 standard deviations above or below the mean were included, and compared to the average RPE cell at the same location. Data are presented as mean ± standard deviation., Results: Within 420 RPE cells examined, 42 cells were further analyzed due to extremes regarding total granule numbers. In addition, 20 RPE cells had AF 1.5 standard deviations below, 28 RPE cells above the mean local AF intensity. Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity. RPE cells with high granule content have nearly twice (1.8 times) as many granules as an average RPE cell., Conclusions: In normal eyes, outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells, suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level. The AF of a cell is related to the composition of intracellular granule types. Ongoing studies using AMD donor eyes will examine possible disease related changes in granule distribution and further put lipofuscińs role in aging and AMD further into perspective., Competing Interests: Conflicts of Interest: The authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/aes-2021-01). The series “Retinal Imaging and Diagnostics” was commissioned by the editorial office without any funding or sponsorship. Dr. Heintzmann reports grants from NIH/NEI 1R01EY027948, during the conduct of the study. Dr. Curcio reports grants from NEI/NIH 1R01EY06109, grants from NEI/NIH 1R01EY027948, during the conduct of the study; grants from Heidelberg Engineering, grants from Genentech/Hoffman LaRoche, other from MacRegen Inc, outside the submitted work. Dr. Sloan reports other from MacRegen, outside the submitted work. Dr. Ach reports grants from NIH/NEI 1R01EY027948, grants from Dr Werner Jackstädt Foundation, other from MacRegen, during the conduct of the study. The authors have no other conflicts of interest to declare.

Purpose: Quantitative fundus autofluorescence (QAF) enables comparisons of autofluorescence intensities among participants. While clinical QAF reports mostly focused on the healthy and diseased adult retina, only very limited data of QAF in the maturing eye are available. Here, we report QAF in a large cohort of healthy children., Methods: In this prospective monocentric cross-sectional study, 70 healthy Caucasian children (5-18 years) were multimodal imaged, including QAF and spectral domain optical coherence tomography. QAF and retinal thicknesses were measured at predefined locations (along horizontal meridian; Early Treatment Diabetic Retinopathy Study [ETDRS] grid) and correlated using custom written Fiji plugins. Standard retinae for different age groups were generated., Results: Fifty-three participants were included. QAF was low in childhood but increased steadily (P < 0.001), also at the fovea (P < 0.001), with no gender differences (P = 0.61). The QAF distribution was similar to adults showing highest values superior-temporally. At individual points, retinal thickness remained stable, while using the ETDRS pattern, the retinal pigment epithelium (RPE) thickness increased significantly with aging. Standard QAF retinae of age groups also showed an increase with aging., Conclusions: QAF can be reliably performed in young children. Function-structure correlation showed a thickening of the RPE and an increasing QAF with aging, probably related to the histologic low number of RPE autofluorescent granules at a younger age but further deposition of these granules during maturation. Standard retinae help to distinguish abnormal QAF in the diseased retina of age-matched patients., Translational Relevance: Our data bridge the gap between preclinical QAF and clinical data application and structural OCT correlation in children.

Purpose: To refine estimates of macular soft drusen abundance in eyes with age-related macular degeneration (AMD) and evaluate hypotheses about drusen biogenesis, we investigated topographic distribution and growth rates of drusen by optical coherence tomography (OCT). We compared results to retinal features with similar topographies (cone density and macular pigment) in healthy eyes., Methods: In a prospective study, distribution and growth rates of soft drusen in eyes with AMD were identified by human observers in OCT volumes and analyzed with computer-assistance. Published histologic data for macular cone densities (n = 12 eyes) and in vivo macular pigment optical density (MPOD) measurements in older adults with unremarkable maculae (n = 31; 62 paired eyes, averaged) were revisited. All values were normalized to Early Treatment Diabetic Retinopathy Study (ETDRS) subfield areas., Results: Sixty-two eyes of 44 patients were imaged for periods up to 78 months. Soft drusen volume per unit volume at baseline is 24.6-fold and 2.3-fold higher in the central ETDRS subfield than in outer and inner rings, respectively, and grows most prominently there. Corresponding ratios (central versus inner and central versus outer) for cone density in donor eyes is 13.3-fold and 5.1-fold and for MPOD, 24.6 and 23.9-fold, and 3.6 and 3.6-fold., Conclusions: Normalized soft drusen volume in AMD eyes as assessed by OCT is ≥ 20-fold higher in central ETDRS subfields than in outer rings, paralleling MPOD distribution in healthy eyes. Data on drusen volume support this metric for AMD risk assessment and clinical trial outcome measure. Alignment of different data modalities support the ETDRS grid for standardizing retinal topography in mechanistic studies of drusen biogenesis.

Purpose: Hyperreflective foci (HRF) are OCT biomarkers for the progression of nonneovascular age-related macular degeneration (AMD) attributed to anteriorly migrated retinal pigment epithelial cells. We examined associations between rod- and cone-mediated vision and HRF plus smaller hyperreflective specks (HRS); we identified a histologic candidate for HRS., Design: Cross-sectional study and histologic survey., Participants: Patients with healthy maculae (n = 34), early AMD (n = 26), and intermediate AMD (n = 41)., Methods: AMD severity was determined by color fundus photography. In OCT scans, HRF and HRS were counted manually. Vision tests probed cones (best-corrected visual acuity [VA], contrast sensitivity), mixed cones and rods (low-luminance VA, low-luminance deficit, mesopic light sensitivity), or rods (scotopic light sensitivity, rod-mediated dark adaptation [RMDA]). An online AMD histopathologic resource was reviewed., Main Outcome Measures: Vision in eyes assessed for HRF and HRS; histologic candidate for HRS., Results: In 101 eyes of 101 patients, HRF and HRS were identified in 25 and 95 eyes, respectively, with good reliability. Hyperreflective foci were present but sparse in healthy eyes, infrequent in early AMD eyes, and frequent but highly variable among intermediate AMD eyes (mean±standard deviation [SD] number per eye, 0.1 ± 0.2, 0.2 ± 0.5, and 1.9 ± 3.4 for healthy, early AMD, and intermediate AMD eyes, respectively). Hyperreflective specks outnumbered HRF in all groups (mean±SD, 4.5 ± 3.2, 6.3 ± 5.8, and 19.4 ± 22.4, respectively). Delayed RMDA was associated strongly with more HRF and HRS (P < 0.0001). Hyperreflective foci also were associated with worse low-luminance VA (P = 0.0117). Hyperreflective specks were associated with worse contrast sensitivity (P = 0.0278), low-luminance VA (P = 0.0010), low-luminance deficit (P = 0.0031), and mesopic (P = 0.0018) and scotopic (P < 0.0001) sensitivity. By histologic analysis, cone lipofuscin was found in outer retinal layers of 25% of healthy aged eyes., Conclusions: Hyperreflective foci and HRS are markers of cellular activity associated with visual dysfunction, especially delayed RMDA, an AMD risk indicator assessing efficiency of retinoid resupply. Hyperreflective specks may represent lipofuscin translocating inwardly within cones. HRF and HRS may serve as structural end points in clinical trials targeting AMD stages earlier than atrophy expansion. These results should be confirmed in a larger sample., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the effect of systemic chloroquine/hydroxychloroquine (CQ/HCQ) on outer retinal health using quantitative fundus autofluorescence (QAF) imaging., Methods: For this prospective, cross-sectional study, 44 CQ/HCQ patients and 25 age-matched controls underwent multimodal retinal imaging including QAF (488 nm) and spectral-domain optical coherence tomography (SD-OCT) in addition to the recommended CQ/HCQ screening procedures. Custom written FIJI plugins enabled detailed QAF analysis and correlation with retinal thickness and comparison to the healthy controls., Results: Out of 44 patients, 29 (mean age 43.5 ± 12.2, range 22-59 years) exposed to CQ/HCQ (mean cumulative dose 724.2 ± 610.4 g, median 608.0 g, range 18.6-2171.0 g) met eligibility criteria. Four of these 29 patients had bull's-eye maculopathy (BEM). Mean QAF values were significantly higher in CQ/HCQ patients than in healthy controls. QAF increase started early after treatment onset, remained high even years after treatment cessation, and was not accompanied by pathologies in the other screening methods, including retinal thicknesses (except in BEM patients)., Conclusions: QAF might be a useful tool in retinal imaging and in verifying systemic CQ/HCQ intake. The early onset and preserved high levels of QAF parallel findings of CQ deposition in the retina in animal models. Whether QAF can be used as a screening tool to detect early CQ/HCQ related maculopathy is the subject of long-term ongoing studies., Translation Relevance: Experimental QAF imaging in systemic CQ/HCQ therapy monitoring might be a useful tool to indicate the drug or its metabolites and to detect metabolic retinal changes., Competing Interests: Disclosure: C. Reichel, None; A. Berlin, None; V. Radun, None; I.-S. Tarau, None; J. Hillenkamp, None; N. Kleefeldt, None; K.R. Sloan, MacRegen (I); T. Ach, Novartis (F, R), Roche (C), MacRegen (I), (Copyright 2020 The Authors.)

Purpose: To determine the abundance and multimodal visibility of drusen and basal linear deposit (BLinD) in early age-related macular degeneration., Methods: A 69-year-old white man was imaged by color fundus photography and red free photography, fundus autofluorescence, and optical coherence tomography. From en face images, we determined the drusen field, drusen area, and equivalent diameters of individual drusen. From high-resolution light-microscopic histology (6 months after the last clinic visit), we determined the area of drusen, BLinD, and pre-BLinD in a subretinal pigment epithelium-basal lamina lipid field., Results: In right and left eyes, respectively, BLinD covered 40% and 46% of the lipid field, versus 21% and 14% covered by drusen. The lipid field was covered 60% to 61% by Drusen + BLinD and 65% to 72% by BLinD + pre-BLinD. In the left eye, the drusen area on color fundus photography (0.18 mm) and red free (0.28 mm) was smaller than the drusen area on histology (1.16 mm). Among drusen confirmed by optical coherence tomography, 55.1% and 56.6% were observed on red free and fundus autofluorescence, respectively., Conclusion: Basal linear deposit covered 1.9 and 3.4-fold more fundus area than soft drusen, silently increasing progression risk. Improved visualization of BLinD and readouts of the retinal pigment epithelium health over lipid will assist population surveillance, early detection, and trial outcome measures.

Purpose: To estimate macular pigment optical density (MPOD) values across different age groups in the South Indian population across various spatial profiles using dual-wavelength autofluorescence., Methods: Sixty eyes of 31 healthy subjects underwent MPOD measurement with Spectralis HRA+OCT. The average MPOD and macular pigment optical volume (MPOV) at 1°, 2°, and 6° radii, the mean MPOD in the classical Early Treatment Diabetic Retinopathy Study (ETDRS) grid, and the spatial profiles of two different age groups across 12 plots covering the radial sectors were recorded., Results: The mean age was 39.1 ± 12.7 years. The mean MPOD and MPOV values were 0.38 ± 0.11 and 787.95 ± 225.13 at 1° eccentricity, 0.23 ± 0.08 and 2000 ± 708.24 at 2° eccentricity, and 0.05 ± 0.02 and 4335 ± 2007.71 at 6° eccentricity, respectively. In the ETDRS grid, the mean MPOD was found to be highest in the central sector and lowest in the inferior peripheral ring. We also found that along the radial sectors the lower quadrants tended to have low MPOD as compared to the upper quadrants. Subjects 40 years of age or older had significantly higher averaged MPOD in certain areas (-15° to 15° and 75° to 105°) along the radial sectors than subjects less than 40 years of age., Conclusions: This study establishes a reference value for future studies of diseased eyes in the South Indian population., Translational Relevance: Our study is unique in that it reports MPOD among the South Indian population across different age groups, as well as the distribution of MPOD in all nine zones of the classical ETDRS grid and various spatial profiles covering the 30° radial sectors centered on the fovea., Competing Interests: Disclosure: R. Srinivasan, None; M.M. Teussink, Heidelberg Engineering GmbH (E); K.R. Sloan, None; J. Surya, None; R. Raman, None, (Copyright 2020 The Authors.)

Purpose: We assessed the association between the abundance of macular xanthophyll carotenoid pigment using dual-wavelength autofluorescence and multimodal vision testing including rod-mediated dark adaptation (RMDA), a measure of retinoid re-supply, in adults ≥60 years old with and without age-related macular degeneration (AMD)., Methods: AMD severity was determined using the nine-step Age-Related Eye Disease Study grading. Tests probed cones (best-corrected visual acuity, contrast sensitivity), cones and rods (low-luminance visual acuity, low-luminance deficit, mesopic light sensitivity), or rods only (scotopic light sensitivity, RMDA). Signal attenuation by macular pigment optical density (MPOD) was estimated using a ratio of blue and green autofluorescence signal to yield mean MPOD in a 1°-diameter fovea-centered disk, mean MPOD in a 2°-diameter disk centered on a perifoveal RMDA test location, and macular pigment optical volume (MPOV, or integrated MPOD) in a 4°-diameter fovea-centered disk. Age-adjusted associations between vision and imaging measures were determined., Results: In 88 eyes of 88 subjects (age, 74.9 ± 5.8 years) with normal eyes (n = 32), early AMD (n = 23), or intermediate AMD (n = 33), foveal and perifoveal MPOD and MPOV were higher in the AMD eyes than in the normal eyes. At the RMDA test location, higher MPOD was unrelated to AMD severity but was associated with faster RMDA., Conclusions: In older adults with and without AMD, higher macular xanthophyll concentrations are associated with better best-corrected visual acuity and RMDA. Data are consistent with a model of cone resilience and rod vulnerability in aging and AMD and can be further explored in a larger sample study.

Purpose: Type 1 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD) may sustain hypoxic and micronutrient-insufficient outer retinal cells compensatorily. We explored this hypothesis via histologic analysis of an eye with a shallow irregular retinal pigment epithelial elevation (SIRE) on OCT and good vision., Design: Case study and clinicopathologic correlation., Participant: A white woman with untreated nonexudative neovascular AMD and 20/30 visual acuity (left eye) and neovascular AMD (right eye), with 9 years' multimodal imaging before dying at 90 years of age., Methods: The left eye was preserved 6.25 hours after death and prepared for submicrometer epoxy resin sections and transmission electron microscopy aligned to clinical OCT B-scans. Inside and outside the MNV area, layer thicknesses, phenotypes, and vascular density of native choriocapillaris and neovessels were measured. Lengths of choriocapillaries and intervening gaps in the index eye and in early AMD eyes and healthy eyes with similar age (n = 19 each) from the Project MACULA (Maculopathy Unveiled by Laminar Analysis) online histopathologic resource (http://projectmacula.cis.uab.edu/) were measured with custom software (Caps and Gaps)., Main Outcome Measures: Descriptive features, vascular density, histologic and OCT layer thicknesses, and distribution of choriocapillaries and intervening gaps., Results: The SIRE correlated to a type 1 MNV that expanded slowly without evidence of exudation and with numerous choroidal vessels traversing Bruch's membrane defects, some visible on OCT. Tissue layers in and adjacent to the MNV area showed continuous RPE and characteristic AMD deposits. Capillary-like neovessels with fenestrations and caveolae resembling native choriocapillaris lined the retinal pigment epithelium (RPE) with a vascular density comparable with surrounding non-MNV areas. Relative to early AMD and healthy aged eyes, the index eye showed similar capillary lengths but larger gaps between vessels, indicating dropout. Outer nuclear layer thickness was preserved and showed less photoreceptor degeneration over areas of relative choriocapillaris health, including the type 1 MNV., Conclusions: Eyes with nonexudative type 1 MNV in AMD may progress to exudation, yet this stable MNV complex supported outer retinal structure for 9 years. Distinguishing features were numerous connecting vessels, high density of neovessels, continuous RPE, and slow growth. Maintaining beneficial type 1 MNV may be a therapeutic strategy., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

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.

Purpose: To use multimodal retinal images (including quantitative fundus autofluorescence [QAF]) for spectral-domain optical coherence tomography (SD-OCT)-based image registration and alignment. For each age decade of healthy adults, normative fine-grained QAF retinal maps are generated and advanced methods for QAF image analysis are applied., Methods: Multimodal retinal images were obtained from 103 healthy subjects (age 19-77 years; unremarkable retina/macula, age-appropriate clear optic media). Custom written FIJI plugins enabled: (1) determination of the fovea in SD-OCT and the edge of the optic disc in infrared (IR) images; (2) alignment and superimposition of multimodal retinal images based on foveal and optic disc position; (3) plotting of normative QAF retinal maps for each decade; and (4) comparison of individual retinas with normative retinas of different decades using newly introduced analysis patterns (QAF97, freehand tool)., Results: SD-OCT based image registration enables easy image registration, alignment, and analysis of different modalities (QAF, IR, and SD-OCT here reported). In QAF, intensities significantly increase with age with two major inclines between the third/fourth and seventh/eighth decades. With aging, the parafoveal area of maximum QAF intensity slightly shifts from temporal-superior to temporal. Compared with standard QAF analysis, refined QAF analysis patterns reveal a more detailed analysis of QAF, especially in the diseased retina., Conclusions: Age-related QAF normative retinal maps can be used to directly compare and classify individual's QAF intensities. Advanced QAF analysis tools will further help to interpret autofluorescence changes in normal aging and in the diseased retina in a multimodal imaging setting., Translational Relevance: Advanced methods for QAF analysis link basic findings with clinical observations in normal aging and in the diseased macula., Competing Interests: Disclosure: N. Kleefeldt, None; K. Bermond, None; I.-S. Tarau, None; J. Hillenkamp, None; A. Berlin, None; K.R. Sloan, MacRegen (I); T. Ach, Novartis (F, R), Roche (C), MacRegen (I), (Copyright 2020 The Authors.)

Background: Age-related macular degeneration (AMD), a leading cause of irreversible vision impairment in the United States and globally, is a disease of the photoreceptor support system involving the retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris in the setting of characteristic extracellular deposits between outer retinal cells and their blood supply. Research has clearly documented the selective vulnerability of rod photoreceptors and rod-mediated (scotopic) vision in early AMD, including delayed rod-mediated dark adaptation (RMDA) and impaired rod-mediated light and pattern sensitivity. The unifying hypothesis of the Alabama Study on Early Macular Degeneration (ALSTAR2) is that early AMD is a disease of micronutrient deficiency and vascular insufficiency, due to detectable structural changes in the retinoid re-supply route from the choriocapillaris to the photoreceptors. Functionally this is manifest as delayed rod-mediated dark adaptation and eventually as rod-mediated visual dysfunction in general., Methods: A cohort of 480 older adults either in normal macular health or with early AMD will be enrolled and followed for 3 years to examine cross-sectional and longitudinal associations between structural and functional characteristics of AMD. Using spectral domain optical coherence tomography, the association between (1) subretinal drusenoid deposits and drusen, (2) RPE cell bodies, and (3) the choriocapillaris' vascular density and rod- and cone-mediated vision will be examined. An accurate map and timeline of structure-function relationships in aging and early AMD gained from ALSTAR2, especially the critical transition from aging to disease, will identify major characteristics relevant to future treatments and preventative measures., Discussion: A major barrier to developing treatments and prevention strategies for early AMD is a limited understanding of the temporal interrelationships among structural and functional characteristics while transitioning from aging to early AMD. ALSTAR2 will enable the development of functionally valid, structural biomarkers for early AMD, suitable for use in forthcoming clinical trials as endpoint/outcome measures. The comprehensive dataset will also allow hypothesis-testing for mechanisms that underlie the transition from aging to AMD, one of which is a newly developed Center-Surround model of cone resilience and rod vulnerability., Trial Registration: ClinicalTrials.gov Identifier NCT04112667, October 7, 2019.

Purpose: The human retinal pigment epithelium (RPE) accumulates granules significant for autofluorescence imaging. Knowledge of intracellular accumulation and distribution is limited. Using high-resolution microscopy techniques, we determined the total number of granules per cell, intracellular distribution, and changes related to retinal topography and age., Methods: RPE cells from the fovea, perifovea, and near-periphery of 15 human RPE flat mounts were imaged using structured illumination microscopy (SIM) and confocal fluorescence microscopy in young (≤51 years, n = 8) and older (>80 years, n = 7) donors. Using custom FIJI plugins, granules were marked with computer assistance, classified based on morphological and autofluorescence properties, and analyzed with regard to intracellular distribution, total number per cell, and granule density., Results: A total of 193,096 granules in 450 RPE cell bodies were analyzed. Based on autofluorescence properties, size, and composition, the RPE granules exhibited nine different phenotypes (lipofuscin, two; melanolipofuscin, five; melanosomes, two), distinguishable by SIM. Overall, lipofuscin (low at the fovea but increases with eccentricity and age) and melanolipofuscin (equally distributed at all three locations with no age-related changes) were the major granule types. Melanosomes were under-represented due to suboptimal visualization of apical processes in flat mounts., Conclusions: Low lipofuscin and high melanolipofuscin content within foveal RPE cell bodies and abundant lipofuscin at the perifovea suggest a different genesis, plausibly related to the population of overlying photoreceptors (fovea, cones only; perifovea, highest rod density). This systematic analysis provides further insight into RPE cell and granule physiology and links granule load to cell autofluorescence, providing a subcellular basis for the interpretation of clinical fundus autofluorescence.

Purpose: Lipofuscin and melanolipofuscin organelles in retinal pigment epithelium (RPE) cells are signal sources for clinical fundus autofluorescence (AF). To elucidate the subcellular basis of AF imaging, we identified, characterized, and quantified the frequency of RPE morphology and AF phenotypes in donor eyes with age-related macular degeneration (AMD)., Methods: In 25 RPE-Bruch's membrane flat mounts from 25 eyes, we analyzed 0.4-μm z-stack epifluorescence images of RPE stained with phalloidin for actin cytoskeleton. Using a custom ImageJ plugin, we classified cells selected in a systematic unbiased fashion in six phenotypes representing increasing degrees of pathology. For each cell, area, AF intensity, and number of Voronoi neighbors were compared with phenotype 1 (uniform AF, polygonal morphology) via generalized estimating equations. We also analyzed each cell's neighborhood., Results: In 29,323 cells, compared with phenotype 1, all other phenotypes, in order of increasing pathology, had significantly larger area, reduced AF, and more variable number of neighbors. Neighborhood area and AF showed similar, but subtler, trends. Cells with highly autofluorescent granule aggregates are no more autofluorescent than others and are in fact lower overall in AF. Pre-aggregates were found in phenotype 1. Phenotype 2, which exhibited degranulation despite normal cytoskeleton, was the most numerous nonhealthy phenotype (16.23%)., Conclusions: Despite aggregation of granules that created hyperAF aggregates within cells, overall AF on a per cell basis decreased with increasing severity of dysmorphia (abnormal shape). Data motivate further development of subcellular resolution in clinical fundus AF imaging and inform an ongoing reexamination of the role of lipofuscin in AMD.

Purpose: To correlate multimodal retinal imaging with high-resolution epoxy resin histologic analysis aligned to in vivo tomograms in a patient with exudative aneurysmal type 1 (AT1) neovascularization and hemorrhage secondary to age-related macular degeneration (AMD)., Design: Case study and clinicopathologic correlation., Participant: An 84-year-old man of European descent with AT1 neovascularization secondary to AMD with a 6-year follow-up with combined antiangiogenic and photodynamic therapy., Methods: Multimodal imaging from each clinic visit, including fluorescein angiography, indocyanine green angiography, and OCT, was correlated with ex vivo OCT and high-resolution histologic images of the donor eye, aligned to the en face images showing hemorrhage and exudation., Main Outcome Measures: Location of the branching vascular network and the aneurysmal vascular dilations in angiography, correlated with histologic findings., Results: Clinically, a hemorrhagic detachment of the retinal pigment epithelium (RPE) in the macular area was associated with an AT1 neovascularization extending near the optic nerve head, where the choroid, which was thin overall, was extremely thin. Resolution of the hemorrhage accompanied by progressive macular atrophy and internal changes in the reflectivity of the RPE detachment were observed. Histologic analysis suggested a physical continuity from a hyalinized choroidal artery to a capillary bed (branching vascular network) in the sub-RPE-basal lamina (BL) space without visualization of aneurysmal dilations., Conclusions: Clinicopathologic correlation of AT1 neovascularization from an intact treated eye with dye-based angiographic and OCT scans supports the proposed nomenclature of AT1 neovascularization over polypoidal choroidal vasculopathy. We described continuity of the sub-RPE-BL branching vascular network with choroidal arteries and histologic correlates of common OCT signatures of neovascular AMD. The thinness of choroid in this patient of European descent contrasts with that reported for Asian populations, in which AT1 neovascularization is associated commonly with pachychoroid disease characteristics. This case reinforces the different manifestations of AT1 neovascularization across and within diverse ethnicities and diseases., (Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To examine the association between macular pigment optical density (MPOD) and rod-mediated dark adaptation (RMDA) in persons ≥60 years old with normal maculas as determined by an accepted color fundus photography grading system., Methods: This cross-sectional analysis used baseline data from eyes in the Alabama Study on Early Age-Related Macular Degeneration. Eyes at step 1 in the AREDS 9-step grading system were considered normal. Eyes were additionally assessed by spectral domain optical coherence tomography (SD-OCT). Foveal MPOD was estimated via heterochromatic flicker photometry, and RMDA was assessed with a computerized dark adaptometer. The association between RMDA and MPOD was examined via Spearman correlation coefficients adjusted for age., Results: In 306 eyes from 306 persons (mean age 68.2 years) in normal macular health, MPOD was not associated with RMDA (age-adjusted rank correlation = 0.043, p = 0.45). After 81 eyes with incidental macular findings by SD-OCT evaluation were excluded, the association between MPOD and RMDA remained null (N = 225, age-adjusted r = 0.015, p = 0.82)., Conclusion: In a large sample of normal aged eyes, RMDA, a visual function that is rate limited by retinoid availability to photoreceptors across the complex of retinal pigment epithelium, Bruch's membrane, and choriocapillaris, is not related to MPOD in the neurosensory retina.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: Drusenoid pigment epithelium detachment (DPED) is a known precursor to geographic atrophy in age-related macular degeneration (AMD). We sought histologic correlates for spectral-domain (SD) optical coherence tomography (OCT) signatures in DPED and determined the frequency and origin of these OCT signatures in a clinical cohort of DPED eyes., Design: Laboratory imaging and histologic comparison, and retrospective, observational cohort study., Participants: Four donor eyes with histopathologic diagnosis of AMD (2 with nonneovascular DPED and 2 with neovascular pigment epithelium detachment [PED]) and 49 eyes of 33 clinic patients with nonneovascular DPED more than 2 mm in diameter., Methods: Donor eyes underwent multimodal ex vivo imaging, including SD OCT, then processing for high-resolution histologic analysis. All clinic patients underwent SD OCT, near-infrared reflectance, and color photography., Main Outcome Measures: Histologic correlates for SD OCT signatures in DPED, estimate of coverage by different retinal pigment epithelium (RPE) phenotypes in the DPED surface; frequency and origin of histologically verified SD OCT signatures in a clinical cohort of DPED eyes, and comparisons of histologic features between neovascular PED and DPED resulting from AMD., Results: Intraretinal and subretinal hyperreflective foci as seen on SD OCT correlated to RPE cells on histologic examination. Hypertransmission of light below the RPE-basal lamina band correlated with dissociated RPE. Subretinal hyperreflective material resulting from acquired vitelliform lesions corresponded to regions of apically expelled RPE organelles. In the clinical cohort, all histologically verified reflectivity signatures were visible and quantifiable. The appearance of intraretinal hyperreflective foci was preceded by thickening of the RPE-basal lamina band. Compared with PEDs associated with neovascular AMD, DPEDs had different crystallization patterns, no lipid-filled cells, and thinner basal laminar deposits., Conclusions: Multiple RPE fates in AMD, including intraretinal cells that are highly prognostic for progression, can be followed and quantified reliably using eye-tracked serial SD OCT. This information may be particularly useful for obtaining an accurate timeline of incipient geographic atrophy in clinic populations and for quantifying anatomic end points and response to therapy in AMD clinical trials., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess outer retinal tubulation (ORT) morphology from spectral-domain optical coherence tomography (SD-OCT) volumes and donor eye histology, analyze ORT reflectivity, and estimate the number of cones surviving in ORT., Methods: In SD-OCT volumes from nine patients with advanced AMD, ORT was analyzed en face and in B-scans. The hyperreflective ORT border in cross-section was delineated and surface area calculated. Reflectivity was compared between ORT types (Closed, Open, Forming, and Branching). A flatmount retina from a donor with neovascular AMD was labeled to visualize the external limiting membrane that delimits ORT and allow measurements of cross-sectional cone area, center-to-center cone spacing, and cone density. The number of cones surviving in ORT was estimated., Results: By en face SD-OCT, ORT varies in complexity and shape. Outer retinal tubulation networks almost always contain Closed cross-sections. Spectral-domain OCT volumes containing almost exclusively Closed ORTs showed no significant direction-dependent differences in hyperreflective ORT border intensity. The surface areas of partial ORT assessed by SD-OCT volumes ranged from 0.16 to 1.76 mm2. From the flatmount retina, the average cross-sectional area of cone inner segments was 49.1 ± 7.9 μm2. The average cone spacing was 7.5 ± 0.6 μm. Outer retinal tubulation cone density was 20,351 cones/mm2. The estimated number of cones in ORT in a macula ranged from 26,399 to 186,833 cones, which is 6% to 44% of the cones present in a healthy macula., Conclusions: These first estimates for cone density and number of cones surviving in ORT suggest that ORT formation considerably distorts the photoreceptor mosaic. Results provide additional insight into the reflectivity characteristics and number of ORT cones observable in living patients by SD-OCT, as cones persist and disease progresses.

The ability to noninvasively image the cone photoreceptor mosaic holds significant potential as a diagnostic for retinal disease. Central to the realization of this potential is the development of sensitive metrics for characterizing the organization of the mosaic. Here we evaluated previously-described and newly-developed (Fourier- and Radon-based) methods of measuring cone orientation in simulated and real images of the parafoveal cone mosaic. The proposed algorithms correlated well across both simulated and real mosaics, suggesting that each algorithm provides an accurate description of photoreceptor orientation. Despite high agreement between algorithms, each performed differently in response to image intensity variation and cone coordinate jitter. The integration property of the Fourier transform allowed the Fourier-based method to be resistant to cone coordinate jitter and perform the most robustly of all three algorithms. Conversely, when there is good image quality but unreliable cone identification, the Radon algorithm performed best. Finally, in cases where the cone coordinate reliability was excellent, the method previously described by Pum and colleagues performed best. These descriptors are complementary to conventional descriptive metrics of the cone mosaic, such as cell density and spacing, and have the potential to aid in the detection of photoreceptor pathology.