Adaptive Optics Optical Coherence Tomography Research Articles

Multi-reference global registration of individual A-lines in adaptive optics optical coherence tomography retinal images.

.Significance: Adaptive optics optical coherence tomography (AO-OCT) technology enables non-invasive, high-resolution three-dimensional (3D) imaging of the retina and promises earlier detection of ocular disease. However, AO-OCT data are corrupted by eye-movement artifacts that must be removed in post-processing, a process rendered time-consuming by the immense quantity of data.Aim: To efficiently remove eye-movement artifacts at the level of individual A-lines, including those present in any individual reference volume.Approach: We developed a registration method that cascades (1) a 3D B-scan registration algorithm with (2) a global A-line registration algorithm for correcting torsional eye movements and image scaling and generating global motion-free coordinates. The first algorithm corrects 3D translational eye movements to a single reference volume, accelerated using parallel computing. The second algorithm combines outputs of multiple runs of the first algorithm using different reference volumes followed by an affine transformation, permitting registration of all images to a global coordinate system at the level of individual A-lines.Results: The 3D B-scan algorithm estimates and corrects 3D translational motions with high registration accuracy and robustness, even for volumes containing microsaccades. Averaging registered volumes improves our image quality metrics up to 22 dB. Implementation in CUDA™ on a graphics processing unit registers a volume in only 10.6 s, 150 times faster than MATLAB™ on a central processing unit. The global A-line algorithm minimizes image distortion, improves regularity of the cone photoreceptor mosaic, and supports enhanced visualization of low-contrast retinal cellular features. Averaging registered volumes improves our image quality up to 9.4 dB. It also permits extending the imaging field of view () and depth of focus () beyond what is attainable with single-reference registration.Conclusions: We can efficiently correct eye motion in all 3D at the level of individual A-lines using a global coordinate system.

Three-dimensional assessment of para- and perifoveal photoreceptor densities and the impact of meridians and age in healthy eyes with adaptive-optics optical coherence tomography (AO-OCT).

An adaptive optics optical coherence tomography (AO-OCT) system is used to assess sixty healthy eyes of thirty subjects (age 22 to 75) to evaluate how the outer retinal layers, foveal eccentricity and age effect the mean cone density. The cone mosaics of different retinal planes (the cone inner segment outer segment junction (IS/OS), the cone outer segment combined with the IS/OS (ISOS+), the cone outer segment tips (COST), and the full en-face plane (FEF)) at four main meridians (superior, nasal, inferior, temporal) and para- and perifoveal eccentricities (ecc 2.5° and 6.5°) were analyzed quantitatively. The mean overall cone density was 19,892/mm2 at ecc 2.5° and 13,323/mm2 at ecc 6.5°. A significant impact on cone density was found for eccentricity (up to 6,700/mm2 between ecc 2.5° and 6.5°), meridian (up to 3,700/mm2 between nasal and superior meridian) and layer (up to 1,400/mm2 between FEF and IS/OS). Age showed only a weak negative effect. These factors as well as inter-individual variability have to be taken into account when comparing cone density measurements between healthy and pathologically changed eyes, as their combined effect on density can easily exceed several thousand cones per mm2 even in parafoveal regions.

Label-free adaptive optics imaging of human retinal macrophage distribution and dynamics

Microglia are resident central nervous system macrophages and the first responders to neural injury. Until recently, microglia have been studied only in animal models with exogenous or transgenic labeling. While these studies provided a wealth of information on the delicate balance between neuroprotection and neurotoxicity within which these cells operate, extrapolation to human immune function has remained an open question. Here we examine key characteristics of retinal macrophage cells in live human eyes, both healthy and diseased, with the unique capabilities of our adaptive optics-optical coherence tomography approach and owing to their propitious location above the inner limiting membrane (ILM), allowing direct visualization of cells. Our findings indicate that human ILM macrophage cells may be distributed distinctly, age differently, and have different dynamic characteristics than microglia in other animals. For example, we observed a macular pattern that was sparse centrally and peaked peripherally in healthy human eyes. Moreover, human ILM macrophage density decreased with age (∼2% of cells per year). Our results in glaucomatous eyes also indicate that ILM macrophage cells appear to play an early and regionally specific role of nerve fiber layer phagocytosis in areas of active disease. While we investigate ILM macrophage cells distinct from the larger sample of overall retinal microglia, the ability to visualize macrophage cells without fluorescent labeling in the live human eye represents an important advance for both ophthalmology and neuroscience, which may lead to novel disease biomarkers and new avenues of exploration in disease progression.

Direct laser writing of a titanium dioxide-laden retinal cone phantom for adaptive optics-optical coherence tomography

The spatial resolution of adaptive optics-optical coherence tomography (AO-OCT) enables visualization of retinal components (e.g., photoreceptors), which can advance clinical diagnosis of ocular diseases. Currently, however, variability in AO-OCT system performance suggests a need for standardized physical models, or “phantoms”, that replicate the opto-structural properties of retinal components. Here we employ direct laser writing – a two-photon polymerization-based additive manufacturing technology – using a light scattering titanium (IV) dioxide nanoparticle-laden photomaterial to create novel retinal cone outer segment (OS) phantoms. Fabrication results demonstrate effective mimicry of cone OS diameter and spacing at four retinal eccentricities, while AO-OCT experiments demonstrate cone spacings >4 µm can be resolved. These results suggest a promising methodology for biomimetic photoreceptor phantom development for AO-OCT.

Cellular-Scale Imaging of Transparent Retinal Structures and Processes Using Adaptive Optics Optical Coherence Tomography.

High-resolution retinal imaging is revolutionizing how scientists and clinicians study the retina on the cellular scale. Its exquisite sensitivity enables time-lapse optical biopsies that capture minute changes in the structure and physiological processes of cells in the living eye. This information is increasingly used to detect disease onset and monitor disease progression during early stages, raising the possibility of personalized eye care. Powerful high-resolution imaging tools have been in development for more than two decades; one that has garnered considerable interest in recent years is optical coherence tomography enhanced with adaptive optics. State-of-the-art adaptive optics optical coherence tomography (AO-OCT) makes it possible to visualize even highly transparent cells and measure some of their internal processes at all depths within the retina, permitting reconstruction of a 3D view of the living microscopic retina. In this review, we report current AO-OCT performance and its success in visualizing and quantifying these once-invisible cells in human eyes.

Real-time retinal layer segmentation of OCT volumes with GPU accelerated inferencing using a compressed, low-latency neural network.

Segmentation of retinal layers in optical coherence tomography (OCT) is an essential step in OCT image analysis for screening, diagnosis, and assessment of retinal disease progression. Real-time segmentation together with high-speed OCT volume acquisition allows rendering of en face OCT of arbitrary retinal layers, which can be used to increase the yield rate of high-quality scans, provide real-time feedback during image-guided surgeries, and compensate aberrations in adaptive optics (AO) OCT without using wavefront sensors. We demonstrate here unprecedented real-time OCT segmentation of eight retinal layer boundaries achieved by 3 levels of optimization: 1) a modified, low complexity, neural network structure, 2) an innovative scheme of neural network compression with TensorRT, and 3) specialized GPU hardware to accelerate computation. Inferencing with the compressed network U-NetRT took 3.5 ms, improving by 21 times the speed of conventional U-Net inference without reducing the accuracy. The latency of the entire pipeline from data acquisition to inferencing was only 41 ms, enabled by parallelized batch processing. The system and method allow real-time updating of en face OCT and OCTA visualizations of arbitrary retinal layers and plexuses in continuous mode scanning. To the best our knowledge, our work is the first demonstration of an ophthalmic imager with embedded artificial intelligence (AI) providing real-time feedback.

Pseudo-real-time retinal layer segmentation for high-resolution adaptive optics optical coherence tomography.

We present a pseudo-real-time retinal layer segmentation for high-resolution Sensorless Adaptive Optics-Optical Coherence Tomography (SAO-OCT). Our pseudo-real-time segmentation method is based on Dijkstra's algorithm that uses the intensity of pixels and the vertical gradient of the image to find the minimum cost in a geometric graph formulation within a limited search region. It segments six retinal layer boundaries in an iterative process according to their order of prominence. The segmentation time is strongly correlated to the number of retinal layers to be segmented. Our program permits en face images to be extracted during data acquisition to guide the depth specific focus control and depth dependent aberration correction for high-resolution SAO-OCT systems. The average processing times for our entire pipeline for segmenting six layers in a retinal B-scan of 496 × 400 and 240 × 400 pixels are around 25.60 and 13.76 ms, respectively. When reducing the number of layers segmented to only two layers, the time required for a 240 × 400 pixel image is 8.26 ms.

On the axial location of Gunn's dots

PurposeTo determine the axial location of Gunn's dots in the retina. MethodsAdaptive optics scanning laser ophthalmoscopy (AOSLO) images and adaptive optics – optical coherence tomography (AO-OCT) volumes were collected from a region where Gunn's dots were found inferior to the optic disc from a subject determined by clinical examination to be a glaucoma suspect. AO-OCT volumes were also collected along the horizontal and vertical meridians from six healthy subjects and one glaucoma subject to identify and document other occurrences of Gunn's dots. AO-OCT volumes were registered in three-dimensions and averaged. Gunn's dots were segmented, and their volume, area, and diameter were measured. ResultsAll Gunn's dots imaged in this study from all subjects were confined to the inner limiting membrane, neither extending into the vitreous nor into the nerve fiber layer. The size of the dots was highly variable. The measured volume, area, and diameter (mean ± standard deviation) were 1119.9 ± 590.9 μm3, 220.2 ± 105.5 μm2, and 14.3 ± 3.1 μm, the latter within the range as previously published reports. ConclusionsBased upon evidence from this study and others, Gunn's dots are not thought to be Müller cell end-feet or hyalocytes. We hypothesize that they are related to microglia, either as the by-product of their phagocytosis function, or are actual dead ameboid-shaped microglia who have fulfilled their scavenger role in retinal pathology. Further studies are needed in diseased eyes to determine if they have predictive value.

Depth-resolved optimization of a real-time sensorless adaptive optics optical coherence tomography.

Sensorless adaptive optics optical coherence tomography (AO-OCT) is a technology to image retinal tissue with high resolution by compensating ocular aberrations without wavefront sensors. In this Letter, a fast and robust hill-climbing algorithm is developed to optimize five Zernike modes in AO-OCT with a numerical aperture between that of conventional AO and commercial OCT systems. The merit function is generated in real time using graphics processing unit while axially tracking the retinal layer of interest. A new method is proposed to estimate the largest achievable field of view for which aberrations are corrected uniformly in sensorless AO-OCT.

Morphologic and Functional Assessment of Photoreceptors After Macula-Off Retinal Detachment With Adaptive-Optics OCT and Microperimetry

Limited information is available on morphologic and functional regeneration of photoreceptors after retinal detachment (RD) surgery. This observational clinical study compared morphologic and functional changes of cones after vitrectomy for macula-off retinal detachment. Prospective, fellow-eye comparative case series. StudyPopulation: Five eyes after vitrectomy with gas for macula-off retinal detachment (retinal detachment eyes, RDE) and 5 healthy fellow eyes (HFE) of 5 patients (mean age 59.8 years, macula-off duration 0.5days to 5.5days). ObservationProcedures: Eyes were examined with adaptive-optics optical coherence tomography (AO-OCT), spectral-domain OCT (SDOCT), and microperimetry (MP) at 6 (baseline, BL) and 56weeks (follow-up, FUP) after 23 gauge pars plana vitrectomy and SF6 gas tamponade. Eight corresponding regions at foveal eccentricities of 2.5° (ecc 2.5°) and 6.5° (ecc 6.5°) were analyzed in every eye. AO-OCT en face images and SD-OCT B-scans were graded regarding irregularity and loss of photoreceptor signals ranging from none to severe changes. The number of detectable cones at height of the inner-outer segment junction (IS/OS) and cone outer segment tips (COST) was counted manually in AO-OCT images. MP with a custom grid was used to assess retinal sensitivity at these locations. MainOutcomeMeasures: Cone density, cone pattern regularity and signal attenuation, retinal sensitivity. In comparison to HFE, RDE showed highly irregular cone patterns in AO-OCT and irregular outer retinal bands in SDOCT. Despite significant improvement of cone pattern regularity compared to BL (P < .001), 63% of AO images showed remaining cone pattern irregularity and 45.5% of SDOCT B-scans showed severe signal reduction at FUP. In HFE, mean cone density retrieved from IS/OS and COST remained around 20,000/mm2 (ecc 2.5°) and 16,000/mm2 (ecc 6.5°) at BL and FUP. Cone density of RDE was significantly reduced and ranged between 200/mm2 and 15,600/mm2 (P < .001) at BL. Despite improvement at FUP (P < .001), mean cone density at IS/OS and COST was still lower compared to HFE and ranged between 7790 and 9555 cones/mm2 (P < .001). Mean retinal sensitivity of all measured locations remained 18 dB in HFE and was significantly lower in RDE, with 14.30 dB at BL and 14.64 dB at FUP. Both SDOCT grading and microperimetry sensitivity showed strong correlation with AO-OCT grading and cone density (rho values > 0.750). The combination of AO-OCT, SDOCT, and microperimetry is a powerful tool to capture cone regeneration after vitreoretinal surgery. Our study shows that cone morphology and function improve within 56weeks after RD surgery but structural and functional impairment is still present.

In vivo measurement of organelle motility in human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are well known to play a central role in the progression of numerous retinal diseases. Changes in the structure and function of these cells thus may serve as sensitive biomarkers of disease onset. While in vivo studies have focused on structural changes, functional ones may better capture cell health owing to their more direct connection to cell physiology. In this study, we developed a method based on adaptive optics optical coherence tomography (AO-OCT) and speckle field dynamics for characterizing organelle motility in individual RPE cells. We quantified the dynamics in terms of an exponential decay time constant, the time required for the speckle field to decorrelate. Using seven normal subjects, we found the RPE speckle field to decorrelate in about 5 s. This result has two fundamental implications for future clinical use. First, it establishes a path for generating a normative baseline to which motility of diseased RPE cells can be compared. Second, it predicts an AO-OCT image acquisition time that is 36 times faster than used in our earlier report for individuating RPE cells, thus a major improvement in clinical efficacy.

Three-Dimensional Adaptive Optics-Assisted Visualization of Photoreceptors in Healthy and Pathologically Aged Eyes.

To visualize and characterize photoreceptor (PR) layers on a subcellular level in healthy and pathologically aged eyes using adaptive-optics optical coherence tomography technology (AO-OCT). AO-OCT scanning was performed within a 2° × 2° field of view, focused on the PR layer at different retinal regions in healthy aged eyes (n = 32 eyes/16 subjects; 62- to 85-years old), eyes with early to intermediate AMD (n = 16 eyes/8 subjects, 65- to 83-years old), and eyes with advanced nonneovascular AMD (n = 16 eyes/8 subjects, 61- to 84-years old). Areas of interest were determined by AO-fundus camera, which is part of the AO-OCT system and spectral-domain (SD)-OCT. PR integrity was evaluated in AO-OCT en face and cross-sectional images at the level of inner/outer segment junction (IS/OS) and cone/PR end tips (COST/ETPR). AO-OCT in healthy eyes showed clearly distinguishable and regular IS/OS and COST patterns and an inverse relation between cone density and eccentricity. In early to intermediate AMD, PR in between drusen showed more irregular patterns (P < 0.001) and slightly lower PR density (P ≤ 0.002). Drusen caused attenuated and distorted signals and a loss of PR mosaic. In advanced AMD, IS/OS and COST patterns were affected to different degrees between surrounding area, junctional zone, and atrophic area (P < 0.001), ranging from reduced PR densities to total signal loss. AO-OCT imaging allows the three-dimensional visualization of different PR layers in patients with AMD and age-matched healthy subjects. Thereby, AO-OCT provides a unique insight into PR morphology and shows potential to fill the gap between conventional OCT and histologic examination of the retina.

THREE-DIMENSIONAL ANALYSIS OF RETINAL MICROANEURYSMS WITH ADAPTIVE OPTICS OPTICAL COHERENCE TOMOGRAPHY.

To characterize retinal microaneurysms (MAs) in patients with diabetes using adaptive optics optical coherence tomography (AOOCT) and compare details found in AOOCT with those found in commercially available retinal imaging techniques. Patients with diabetes and MA in the macular area were included in this pilot study. The area of interest, identified in standard fluorescein angiography, was imaged using an AO fundus camera and AOOCT. Microaneurysms were characterized in AOOCT (visibility, reflectivity, feeding/draining vessels, and intraretinal location) and compared with findings in AO fundus camera, OCT angiography, and fluorescein angiography. Fifty-three MAs were imaged in 15 eyes of 10 patients. Feeding and/or draining vessels from both capillary plexus could be identified in 34 MAs in AOOCT images. Of 45 MAs imaged with OCT angiography, 18 (40%) were visible in the superior plexus, 12 (27%) in the deep capillary plexus, and 15 MAs (33%) could not be identified at all. Intraluminal hyperreflectivity, commonly seen in AO fundus camera, corresponded only in 8 of 27 cases (30%) to intraluminal densities seen in AOOCT. Adaptive optics OCT imaging revealed that MAs located in the inner nuclear layer were connected to the intermediate and/or deep capillary plexus. Intraluminal hyperreflectivity seen on AO fundus camera images originated from a strong reflection from the vessel wall and only in a third of the cases from intraluminal clots. Currently, AOOCT is the most expedient in vivo imaging method to capture morphologic details of retinal microvasculature in 3D and in the context of the surrounding retinal anatomy.

Local wavefront mapping in tissue using computational adaptive optics OCT.

The identification and correction of wavefront aberrations is often necessary to achieve high-resolution optical images of biological tissues, as imperfections in the optical system and the tissue itself distort the imaging beam. Measuring the localized wavefront aberration provides information on where the beam is distorted and how severely. We have recently developed a method to estimate the single-pass wavefront aberrations from complex optical coherence tomography (OCT) data. Using this method, localized wavefront measurement and correction using computational OCT was performed in ex vivo tissues. The computationally measured wavefront varied throughout the imaged OCT volumes and, therefore, a local wavefront correction outperformed a global wavefront correction. The local wavefront measurement was also used to generate tissue aberration maps. Such aberration maps could potentially be used as a new form of tissue contrast.

Dynamic Changes of Retinal Microaneurysms in Diabetes Imaged With In Vivo Adaptive Optics Optical Coherence Tomography.

To prospectively monitor microaneurysms (MAs) in three dimensions using adaptive optics optical coherence tomography (AOOCT). Patients with diabetes mellitus and parafoveal MAs were included in this longitudinal study. At baseline, MAs were identified in standard fluorescein angiography (FA) and subsequently imaged with an AOOCT prototype, incorporated into an AO fundus camera (RTX1, Imagine Eyes) device. Imaging was repeated every 3 months in each patient to explore the potential structural change of MAs over time including size, shape, intraretinal position, (intra-) luminal reflectivity, and other qualitative morphologic characteristics. We imaged 18 MAs in seven eyes (two left eyes) of five patients (mean age: 69 ± 7 years) over 18 months. All MAs appeared as saccular in the en face imaging plane at baseline, and no change in shape was observed in any of the MAs during follow-up. Evaluation of the AOOCT volumes revealed dynamic changes of MAs during follow-up including intermittent growth (n = 2), progressive involution (n = 3), total disappearance (n = 2), and MA division (n = 1). Intraluminal hyperreflective material was visualized in 11 out of 18 MAs, which remained stable (n = 3), increased (n = 2), regressed (n = 1), or fluctuated (n = 5). Three MAs without intraluminal spots at baseline progressively developed distinct hyperreflectivities. AOOCT illustrates the structurally dynamic evolution of MAs in vivo in three dimensions. Despite a consistent saccular shape in the en face view, AOOCT volumes revealed a heterogeneous behavior in regard to size and reflective status of MAs over time.

Automated identification of cone photoreceptors in adaptive optics optical coherence tomography images using transfer learning

Automated measurements of the human cone mosaic requires the identification of individual cone photoreceptors. The current gold standard, manual labeling, is a tedious process and can not be done in a clinically useful timeframe. As such, we present an automated algorithm for identifying cone photoreceptors in adaptive optics optical coherence tomography (AO-OCT) images. Our approach fine-tunes a pre-trained convolutional neural network originally trained on AO scanning laser ophthalmoscope (AO-SLO) images, to work on previously unseen data from a different imaging modality. On average, the automated method correctly identified 94% of manually labeled cones when compared to manual raters, from twenty different AO-OCT images acquired from five normal subjects. Voronoi analysis confirmed the general hexagonal-packing structure of the cone mosaic as well as the general cone density variability across portions of the retina. The consistency of our measurements demonstrates the high reliability and practical utility of having an automated solution to this problem.

The Properties of Outer Retinal Band Three Investigated With Adaptive-Optics Optical Coherence Tomography.

PurposeOptical coherence tomography's (OCT) third outer retinal band has been attributed to the zone of interdigitation between RPE cells and cone outer segments. The purpose of this paper is to investigate the structure of this band with adaptive optics (AO)-OCT.MethodsUsing AO-OCT, images were obtained from two subjects. Axial structure was characterized by measuring band 3 thickness and separation between bands 2 and 3 in segmented cones. Lateral structure was characterized by correlation of band 3 with band 2 and comparison of their power spectra. Band thickness and separation were also measured in a clinical OCT image of one subject.ResultsBand 3 thickness ranged from 4.3 to 6.4 μm. Band 2 correlations ranged between 0.35 and 0.41 and power spectra of both bands confirmed peak frequencies that agree with histologic density measurements. In clinical images, band 3 thickness was between 14 and 19 μm. Measurements of AO-OCT of interband distance were lower than our corresponding clinical OCT measurements.ConclusionsBand 3 originates from a structure with axial extent similar to a single surface. Correlation with band 2 suggests an origin within the cone photoreceptor. These two observations indicate that band 3 corresponds predominantly to cone outer segment tips (COST). Conventional OCT may overestimate both the thickness of band 3 and outer segment length.

Review of adaptive optics OCT (AO-OCT): principles and applications for retinal imaging [Invited

In vivo imaging of the human retina with a resolution that allows visualization of cellular structures has proven to be essential to broaden our knowledge about the physiology of this precious and very complex neural tissue that enables the first steps in vision. Many pathologic changes originate from functional and structural alterations on a cellular scale, long before any degradation in vision can be noted. Therefore, it is important to investigate these tissues with a sufficient level of detail in order to better understand associated disease development or the effects of therapeutic intervention. Optical retinal imaging modalities rely on the optical elements of the eye itself (mainly the cornea and lens) to produce retinal images and are therefore affected by the specific arrangement of these elements and possible imperfections in curvature. Thus, aberrations are introduced to the imaging light and image quality is degraded. To compensate for these aberrations, adaptive optics (AO), a technology initially developed in astronomy, has been utilized. However, the axial sectioning provided by retinal AO-based fundus cameras and scanning laser ophthalmoscope instruments is limited to tens of micrometers because of the rather small available numerical aperture of the eye. To overcome this limitation and thus achieve much higher axial sectioning in the order of 2-5µm, AO has been combined with optical coherence tomography (OCT) into AO-OCT. This enabled for the first time in vivo volumetric retinal imaging with high isotropic resolution. This article summarizes the technical aspects of AO-OCT and provides an overview on its various implementations and some of its clinical applications. In addition, latest developments in the field, such as computational AO-OCT and wavefront sensor less AO-OCT, are covered.

Photoreceptor disc shedding in the living human eye.

Cone photoreceptors undergo a daily cycle of renewal and shedding of membranous discs in their outer segments (OS), the portion responsible for light capture. These physiological processes are fundamental to maintaining photoreceptor health, and their dysfunction is associated with numerous retinal diseases. While both processes have been extensively studied in animal models and postmortem eyes, little is known about them in the living eye, in particular human. In this study, we report discovery of the optical signature associated with disc shedding using a method based on adaptive optics optical coherence tomography (AO-OCT) in conjunction with post-processing methods to track and monitor individual cone cells in 4D. The optical signature of disc shedding is characterized by an abrupt transient loss in the cone outer segment tip (COST) reflection followed by its return that is axially displaced anteriorly. Using this signature, we measured the temporal and spatial properties of shedding events in three normal subjects. Average duration of the shedding event was 8.8 ± 13.4 minutes, and average length loss of the OS was 2.1 μm (7.0% of OS length). Prevalence of cone shedding was highest in the morning (14.3%) followed by the afternoon (5.7%) and evening (4.0%), with load distributed across the imaged patch. To the best of our knowledge these are the first images of photoreceptor disc shedding in the living retina.

3D Imaging of Retinal Pigment Epithelial Cells in the Living Human Retina.

PurposeDysfunction of the retinal pigment epithelium (RPE) underlies numerous retinal pathologies, but biomarkers sensitive to RPE change at the cellular level are limited. In this study, we used adaptive optics optical coherence tomography (AO-OCT) in conjunction with organelle motility as a novel contrast mechanism to visualize RPE cells and characterize their 3-dimensional (3D) reflectance profile.MethodsUsing the Indiana AO-OCT imaging system (λc = 790 nm), volumes were acquired in the macula of six normal subjects (25–61 years). Volumes were registered in 3D with subcellular accuracy, layers segmented, and RPE and photoreceptor en face images extracted and averaged. Voronoi and two-dimensional (2D) power spectra analyses were applied to the images to quantify RPE and cone packing and cone-to-RPE ratio.ResultsAdaptive optics OCT revealed two distinct reflectance patterns at the depth of the RPE. One is characterized by the RPE interface with rod photoreceptor tips, the second by the RPE cell nuclei and surrounding organelles, likely melanin. Increasing cell contrast by averaging proved critical for observing the RPE cell mosaic, successful in all subjects and retinal eccentricities imaged. Retinal pigment epithelium mosaic packing and cell thickness generally agreed with that of histology and in vivo studies using other imaging modalities.ConclusionsWe have presented, to our knowledge, the first detailed characterization of the 3D reflectance profile of individual RPE cells and their relation to cones and rods in the living human retina. Success in younger and older eyes establishes a path for testing aging effects in larger populations. Because the technology is based on OCT, our measurements will aid in interpreting clinical OCT images.