Long-term mesoscale imaging of 3D intercellular dynamics across a mammalian organ.

A comprehensive understanding of physio-pathological processes necessitates non-invasive intravital three-dimensional (3D) imaging over varying spatial and temporal scales. However, huge data throughput, optical heterogeneity, surface irregularity, and phototoxicity pose great challenges, leading to an inevitable trade-off between volume size, resolution, speed, sample health, and system complexity. Here, we introduce a compact real-time, ultra-large-scale, high-resolution 3D mesoscope (RUSH3D), achieving uniform resolutions of 2.6 × 2.6 × 6 µm3 across a volume of 8,000 × 6,000 × 400 µm3 at 20 Hz with low phototoxicity. Through the integration of multiple computational imaging techniques, RUSH3D facilitates a 13-fold improvement in data throughput and an orders-of-magnitude reduction in system size and cost. With these advantages, we observed premovement neural activity and cross-day visual representational drift across the mouse cortex, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and heterogeneous immune responses following traumatic brain injury-all at single-cell resolution, opening up a horizon for intravital mesoscale study of large-scale intercellular interactions at the organ level.

Characterizing Vascular Wall and Lumen Caliber in Eyes with Diabetic Retinopathy Based on Adaptive Optics Scanning Laser Ophthalmoscopy.

(200/200) Purpose: Our aim was to evaluate structural alterations of retinal arterioles due to type 1 diabetes (T1D) and/or diabetic retinopathy (DR) under AOSLO imaging. METHODS: Each study eye underwent mydriasis and AOSLO imaging in a single-visit study. The instrument's arrangement of four offset aperture images provided two orthogonal split-detector images and enabled isotropic analysis of the arteriolar boundaries. For each arteriole, we calculated the wall-to-lumen ratio (WLR), mean wall thickness, and luminal and external diameters. RESULTS: In total, we enrolled 5 (20.8%) healthy control eyes and 19 eyes of patients with T1D. The DR distribution was: four (16.7%) no-DR, nine (37.5%%) mild or moderate nonproliferative DR (NPDR), and six (25%) severe NPDR or proliferative DR. Mean wall thickness increased significantly in eyes with T1D compared to healthy controls (p = 0.0006) and in eyes with more advanced DR (p = 0.0004). The WLR was significantly higher in eyes with T1D (p = 0.002) or more severe DR (p = 0.004). There was no significant relationship between T1D status or DR severity and any of the arteriolar diameters. CONCLUSIONS: In this preliminary study, there appeared to be increases in the WLR and mean wall thickness in eyes with T1D and more severe DR than in the controls and eyes with no/less severe DR. Future studies may further elucidate the relationship between the retinal arteriolar structure and physiologic alterations in DR.

Neural adaptation to the eye's optics through phase compensation.

How does the brain achieve a seemingly veridical and 'in-focus' perception of the world, knowing how severely corrupted visual information is by the eye's optics? Optical blur degrades retinal image quality by reducing the contrast and disrupting the phase of transmitted signals. Neural adaptation can attenuate the impact of blur on image contrast, yet vision rather relies on perceptually-relevant information contained within the phase structure of natural images. Here we show that neural adaptation can compensate for the impact of optical aberrations on phase congruency. We used adaptive optics to fully control optical factors and test the impact of specific optical aberrations on the perceived phase of compound gratings. We assessed blur-induced changes in perceived phase over three distinct exposure spans. Under brief blur exposure, perceived phase shifts matched optical theory predictions. During short-term (~1h) exposure, we found a reduction in blur-induced phase shifts over time, followed by after-effects in the opposite direction-a hallmark of adaptation. Finally, patients with chronic exposure to poor optical quality showed altered phase perception when tested under fully-corrected optical quality, suggesting long-term neural compensatory adjustments to phase spectra. These findings reveal that neural adaptation to optical aberrations compensates for alterations in phase congruency, helping restore perceptual quality over time.

Macular vascular and photoreceptor changes for diabetic macular edema at early stage.

This study was intended to investigate the macular vascular and photoreceptor changes for diabetic macular edema (DME) at the early stage. A total of 255 eyes of 134 diabetes mellitus patients were enrolled and underwent an ophthalmological and systemic evaluation in this cross-sectional study. Early DME was characterized by central subfoveal thickness (CST) value between 250 and 325 µm, intact ellipsoid zone, and an external limiting membrane. While non-DME was characterized by CST < 250 µm with normal retinal morphology and structure. Foveal avascular zone (FAZ) area ≤ 0.3 mm2 (P < 0.001, OR = 0.41, 95% CI 0.26-0.67 in the multivariate analysis) and HbA1c level ≤ 8% (P = 0.005, OR = 0.37, 95% CI 0.19-0.74 in multivariate analysis) were significantly associated with a higher risk of early DME. Meanwhile, no significant differences exist in cone parameters between non-DME and early DME eyes. Compared with non-DME eyes, vessel diameter, vessel wall thickness, wall-to-lumen ratio, the cross-sectional area of the vascular wall in the upper side were significantly decreased in the early DME eyes (P = 0.001, P < 0.001, P = 0.005, P = 0.003 respectively). This study suggested a vasospasm or vasoconstriction with limited further photoreceptor impairment at the early stage of DME formation. CST ≥ 250 µm and FAZ ≤ 0.3 mm2 may be the indicator for early DME detection.

Controlling cantilevered adaptive X-ray mirrors.

Modeling the behavior of a prototype cantilevered X-ray adaptive mirror (held from one end) demonstrates its potential for use on high-performance X-ray beamlines. Similar adaptive mirrors are used on X-ray beamlines to compensate optical aberrations, control wavefronts and tune mirror focal distances at will. Controlled by 1D arrays of piezoceramic actuators, these glancing-incidence mirrors can provide nanometre-scale surface shape adjustment capabilities. However, significant engineering challenges remain for mounting them with low distortion and low environmental sensitivity. Finite-element analysis is used to predict the micron-scale full actuation surface shape from each channel and then linear modeling is applied to investigate the mirrors' ability to reach target profiles. Using either uniform or arbitrary spatial weighting, actuator voltages are optimized using a Moore-Penrose matrix inverse, or pseudoinverse, revealing a spatial dependence on the shape fitting with increasing fidelity farther from the mount.

Compact Linear Flow Phantom Model for Retinal Blood-Flow Evaluation.

Impaired retinal blood flow is associated with ocular diseases such as glaucoma, macular degeneration, and diabetic retinopathy. Among several ocular imaging techniques developed to measure retinal blood flow both invasively and non-invasively, adaptive optics (AO)-enabled scanning laser ophthalmoscopy (AO-SLO) resolves individual red blood cells and provides a high resolution with which to measure flow across retinal microvasculature. However, cross-validation of flow measures remains a challenge owing to instrument and patient-specific variability in each imaging technique. Hence, there is a critical need for a well-controlled clinical flow phantom for standardization and to establish blood-flow measures as clinical biomarkers for early diagnosis. Here, we present the design and validation of a simple, compact, portable, linear flow phantom based on a direct current motor and a conveyor-belt system that provides linear velocity tuning within the retinal microvasculature range (0.5-7 mm/s). The model was evaluated using a sensitive AO-SLO line-scan technique, which showed a <6% standard deviation from the true velocity. Further, a clinical SLO instrument showed a linear correlation with the phantom's true velocity (r2 > 0.997). This model has great potential to calibrate, evaluate, and improve the accuracy of existing clinical imaging systems for retinal blood flow and aid in the diagnosis of ocular diseases with abnormal blood flow.

Minimum intensity projection of embossed quadrant-detection images for improved photoreceptor mosaic visualisation.

Non-confocal split-detection imaging reveals the cone photoreceptor inner segment mosaic in a plethora of retinal conditions, with the potential of providing insight to ageing, disease, and response to treatment processes, in vivo, and allows the screening of candidates for cell rescue therapies. This imaging modality complements confocal reflectance adaptive optics scanning light ophthalmoscopy, which relies on the waveguiding properties of cones, as well as their orientation toward the pupil. Split-detection contrast, however, is directional, with each cone inner segment appearing as opposite dark and bright semicircles, presenting a challenge for either manual or automated cell identification. Quadrant-detection imaging, an evolution of split detection, could be used to generate images without directional dependence. Here, we demonstrate how the embossed-filtered quadrant-detection images, originally proposed by Migacz et al. for visualising hyalocytes, can also be used to generate photoreceptor mosaic images with better and non-directional contrast for improved visualisation. As a surrogate of visualisation improvement between legacy split-detection images and the images resulting from the method described herein, we provide preliminary results of simple image processing routines that may enable the automated identification of generic image features, as opposed to complex algorithms developed specifically for photoreceptor identification, in pathological retinas.

Adaptive Optics Optical Coherence Tomography Analysis of Induced Pluripotent Stem Cell-Derived Retinal Organoid Transplantation in Retinitis Pigmentosa.

This study evaluates the transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into patients with advanced retinitis pigmentosa using adaptive optics optical coherence tomography (AO-OCT) to monitor retinal changes over two years post transplantation. Our results confirmed successful engraftment and increased retinal thickness, with AO-OCT providing detailed visualization of cellular structures such as an outer plexiform layer-like line and highly reflective particles within rosette-like formations, indicative of photoreceptor development. Immunohistological analysis in a parallel monkey model confirmed these structures as mature, functional photoreceptor rosettes. The integration of high-resolution AO-OCT with immunohistology provides critical insights into the structural and functional outcomes of transplantation and represents a promising advancement in the treatment of retinal degenerative diseases.

Association of outer retinal and choroidal alterations with neuroimaging and clinical features in posterior cortical atrophy.

BACKGROUND: Posterior cortical atrophy (PCA) is a rare condition characterized by early-onset and progressive visual impairment. Individuals with PCA have relatively early-onset and progressive dementia, posing certain needs for early detection. Hence, this study aimed to investigate the association of alterations in outer retinal and choroidal structure and microvasculature with PCA neuroimaging and clinical features and the possible effects of apolipoprotein E(APOE) ε4 allele on outer retinal and choroidal alterations in participants with PCA, to detect potential ocular biomarkers for PCA screening. METHODS: This cross-sectional study included PCA and age- and sex-matched healthy control participants from June 2022 to December 2023. All participants with PCA completed a comprehensive neurological evaluation. All participants were recorded baseline information and underwent an ophthalmic evaluation. Quantitative analyses were performed using swept-source optical coherence tomography (SS-OCT) and angiography (SS-OCTA). Adaptive optics scanning laser ophthalmoscopy (AO-SLO) was performed in some patients. In participants with PCA, the influence of APOE ε4 on outer retinal and choroidal alterations and the correlation of outer retinal and choroidal alterations with PCA neuroimaging and clinical features in participants with PCA were investigated. RESULTS: A total of 28 participants (53 eyes) with PCA and 56 healthy control participants (112 eyes) were included in the current study. Compared with healthy control participants, participants with PCA had significantly reduced outer retinal thickness (ORT) (p < 0.001), choriocapillaris vessel density (VD) (p = 0.007), choroidal vascular index (CVI) (p = 0.005) and choroidal vascular volume (CVV) (p = 0.003). In participants with PCA, APOE ε4 carriers showed thinner ORT (p = 0.009), and increased choriocapillaris VD (p = 0.004) and CVI (p = 0.004). The PCA neuroimaging features were positively associated with the ORT, CVI and CVV. Furthermore, differential correlations were observed of PCA clinical features with the CRT, CVV and CVI. CONCLUSIONS: Our findings highlighted the association of outer retinal and choroidal alterations with PCA neuroimaging and clinical features in participants with PCA. Noninvasive SS-OCT and SS-OCTA can provide potential biomarkers for the diagnosis and management of PCA, improving awareness of PCA syndrome among ophthalmologists, neurologists, and primary care providers.

Diabetic retinopathy: New concepts of screening, monitoring, and interventions.

The science of diabetes care has progressed to provide a better understanding of the oxidative and inflammatory lesions and pathophysiology of the neurovascular unit within the retina (and brain) that occur early in diabetes, even prediabetes. Screening for retinal structural abnormalities, has traditionally been performed by fundus examination or color fundus photography; however, these imaging techniques detect the disease only when there are sufficient lesions, predominantly hemorrhagic, that are recognized to occur late in the disease process after significant neuronal apoptosis and atrophy, as well as microvascular occlusion with alterations in vision. Thus, interventions have been primarily oriented toward the later-detected stages, and clinical trials, while demonstrating a slowing of the disease progression, demonstrate minimal visual improvement and modest reduction in the continued loss over prolonged periods. Similarly, vision measurement utilizing charts detects only problems of visual function late, as the process begins most often parafoveally with increasing number and progressive expansion, including into the fovea. While visual acuity has long been used to define endpoints of visual function for such trials, current methods reviewed herein are found to be imprecise. We review improved methods of testing visual function and newer imaging techniques with the recommendation that these must be utilized to discover and evaluate the injury earlier in the disease process, even in the prediabetic state. This would allow earlier therapy with ocular as well as systemic pharmacologic treatments that lower the and neuro-inflammatory processes within eye and brain. This also may include newer, micropulsed laser therapy that, if applied during the earlier cascade, should result in improved and often normalized retinal function without the adverse treatment effects of standard photocoagulation therapy.

Multimodal in-vivo maps as a tool to characterize retinal structural biomarkers for progression in adult-onset Stargardt disease.

Purpose: To characterize retinal structural biomarkers for progression in adult-onset Stargardt disease from multimodal retinal imaging in-vivo maps. Methods: Seven adult patients (29-69 years; 3 males) with genetically-confirmed and clinically diagnosed adult-onset Stargardt disease and age-matched healthy controls were imaged with confocal and non-confocal Adaptive Optics Scanning Light Ophthalmoscopy (AOSLO), optical coherence tomography (OCT), fundus infrared (FIR), short wavelength-autofluorescence (FAF) and color fundus photography (CFP). Images from each modality were scaled for differences in lateral magnification before montages of AOSLO images were aligned with en-face FIR, FAF and OCT scans to explore changes in retinal structure across imaging modalities. Photoreceptors, retinal pigment epithelium (RPE) cells, flecks, and other retinal alterations in macular regions were identified, delineated, and correlated across imaging modalities. Retinal layer-thicknesses were extracted from segmented OCT images in areas of normal appearance on clinical imaging and intact outer retinal structure on OCT. Eccentricity dependency in cell density was compared with retinal thickness and outer retinal layer thickness, evaluated across patients, and compared with data from healthy controls. Results: In patients with Stargardt disease, alterations in retinal structure were visible in different image modalities depending on layer location and structural properties. The patients had highly variable foveal structure, associated with equally variable visual acuity (-0.02 to 0.98 logMAR). Cone and rod photoreceptors, as well as RPE-like structures in some areas, could be quantified on non-confocal split-detection AOSLO images. RPE cells were also visible on dark field AOSLO images close to the foveal center. Hypo-reflective gaps of non-waveguiding cones (dark cones) were seen on confocal AOSLO in regions with clinically normal CFP, FIR, FAF and OCT appearance and an intact cone inner segment mosaic in three patients. Conclusion: Dark cones were identified as a possible first sign of retinal disease progression in adult-onset Stargardt disease as these are observed in retinal locations with otherwise normal appearance and outer retinal thickness. This corroborates a previous report where dark cones were proposed as a first sign of progression in childhood-onset Stargardt disease. This also supports the hypothesis that, in Stargardt disease, photoreceptor degeneration occurs before RPE cell death.

Adaptive optics retinal imaging in patients with usher syndrome.

Purpose: To determine the structure of the cone photoreceptor mosaic in the macula in eyes with retinitis pigmentosa related to Usher syndrome using adaptive optics fundus (AO) imaging and to correlate these findings with those of the standard clinical diagnostics. Methods: Ten patients with a genetically confirmed retinitis pigmentosa in Usher syndrome due to biallelic variants in MYO7A or USH2A were enrolled in the study. All patients underwent a complete ophthalmological examination including best corrected visual acuity (BCVA), spectral-domain optical coherence tomography (SD-OCT) with fundus autofluorescence photography (FAF), full-field (ffERG) and multifocal electroretinography (mfERG) and Adaptive Optics Flood Illuminated Ophthalmoscopy (AO, rtx1™, Imagine Eyes, Orsay, France). The cone density was assessed centrally and at each 0.5 degree horizontally and vertically from 1-4 degree of eccentricity. Results: In the AO images, photoreceptor cell death was visualized as a disruption of the cone mosaic and low cone density. In the early stage of the disease, cones were still visible in the fovea, whereas outside the fovea a loss of cones was recognizable by blurry, dark patches. The blurry patches corresponded to the parafoveal hypofluorescent ring in the FAF images and the beginning loss of the IS/OS line and external limiting membrane in the SD-OCT images. FfERGs were non-recordable in 7 patients and reduced in 3. The mfERG was reduced in all patients and correlated significantly (p <0.001) with the cone density. The kinetic visual field area, measured with III4e and I4e, did not correlate with the cone density. Conclusion: The structure of the photoreceptors in Usher syndrome patients were detectable by AO fundus imaging. The approach of using high-resolution technique to assess the photoreceptor structure complements the established clinical examinations and allows a more sensitive monitoring of early stages of retinitis pigmentosa in Usher syndrome.

Intensity-based optoretinography reveals sub-clinical deficits in cone function in retinitis pigmentosa.

Introduction: Clinical tools have been widely used in the diagnosis, description, and monitoring the progression of retinitis pigmentosa (RP); however, many of these methods have inherently low sensitivity and specificity, and significant photoreceptor disruption can occur before RP progression has clinically manifest. Adaptive optics scanning light ophthalmoscopy (AOSLO) has shown promise as a powerful tool for assessing photoreceptor disruption both structurally and functionally due to its increased resolution. Methods: Here we assess photoreceptor structure and function at the cellular level through AOSLO by acquiring intensity based optoretinography (iORG) in 15 individuals with no reported retinal pathology and 7 individuals with a prior clinical diagnosis of RP. Photoreceptor structure was quantified by calculating cone nearest neighbor distance (NND) across different retinal eccentricities from the AOSLO images. Cone outer segment length was measured across different retinal eccentricities using optical coherence tomography (OCT) derived longitudinal reflectivity profiles (LRPs). Finally, iORG measures of photoreceptor function were compared to retinal sensitivity as measured using the macular integrity assessment (MAIA) microperimeter. Results: Broadly, participants with RP exhibited increasing cone nearest neighbor distances and decreasing cone outer segment length as a function of retinal eccentricity, consistent with prior reports for both controls and individuals with RP. Nearly all individuals with RP had reduced iORG amplitudes for all retinal eccentricities when compared to the control cohort, and the reduction was greater in eccentricities further from the fovea. Comparing iORG amplitudes to MAIA retinal sensitivity, we found that the iORG was more sensitive to early changes in photoreceptor function whereas MAIA was more sensitive to later stages of disease. Discussion: This highlights the utility of iORG as a method to detect sub-clinical deficits in cone function in all stages of disease progression and supports the future use of iORG for identifying cells that are candidates for cellular based therapies.

Defining reference values of arterioles in healthy individuals for studies with adaptive optics imaging.

Purpose: To investigate age-dependent wall to lumen ratio (WLR) reference values for healthy individuals in adaptive optics imaging (AO). WLR serves as an objective, dimensionless parameter for the evaluation of structural changes in vessels caused by conditions like arterial hypertension, diabetes or vascular stenosis. Methods: 50 right eyes of healthy individuals were examined by adaptive optics imaging. The central big arterioles and smaller arterial branches at least one disc diameter away from the optic disc, approximately above or below the macula were measured by the manufacturer's software. The wall-lumen-ratio (WLR), the wall cross-sectional area (WCSA) and lumen diameter (LD) were assessed. Subsequent data analysis was performed with a focus on variables including age, gender and blood pressure. Results: Normative values for WLR, WCSA and LD in 5 different age groups could be established. However, no significant differences between the age groups were found. Intra-subject comparisons revealed significantly higher WLRs on peripheral branches when compared to central arterioles. WLR showed in this normotensive cohort no relevant correlation with the systolic, diastolic and mean blood pressure. Gender and intraocular pressure had no influence on the vascular parameters. Conclusion: AO is capable of examining vascular alterations in arterioles at an almost microscopic level. Age did not seem to alter WLR, normotensive blood pressure parameters showed also no significant impact. AO-based vessel analysis may provide clinically useful biomarkers for cardiovascular health and should be tested in future studies.

The effect of sampling window size on topographical maps of foveal cone density.

Purpose: To characterize the effect of sampling window size on maps of foveal cone density derived from adaptive optics scanning light ophthalmoscope (AOSLO) images of the cone mosaic. Methods: Forty-four AOSLO-derived montages of the foveal cone mosaic (300 x 300µm) were used for this study (from 44 individuals with normal vision). Cone photoreceptor coordinates were semi-automatically identified by one experienced grader. From these coordinates, cone density matrices across each foveal montage were derived using 10 different sampling window sizes containing 5, 10, 15, 20, 40, 60, 80, 100, 150, or 200 cones. For all 440 density matrices, we extracted the location and value of peak cone density (PCD), the cone density centroid (CDC) location, and cone density at the CDC. Results: Across all window sizes, PCD values were larger than those extracted at the CDC location, though the difference between these density values decreased as the sampling window size increased (p<0.0001). Overall, both PCD (r=-0.8099, p=0.0045) and density at the CDC (r=-0.7596, p=0.0108) decreased with increasing sampling window size. This reduction was more pronounced for PCD, with a 27.8% lower PCD value on average when using the 200-cone versus the 5-cone window (compared to only a 3.5% reduction for density at the CDC between these same window sizes). While the PCD and CDC locations did not occur at the same location within a given montage, there was no significant relationship between this PCD-CDC offset and sampling window size (p=0.8919). The CDC location was less variable across sampling windows, with an average per-participant 95% confidence ellipse area across the 10 window sizes of 47.56µm² (compared to 844.10µm² for the PCD location, p<0.0001). Conclusion: CDC metrics appear more stable across varying sampling window sizes than PCD metrics. Understanding how density values change according to the method used to sample the cone mosaic may facilitate comparing cone density data across different studies.

Application of novel non-invasive ophthalmic imaging to visualize peripapillary wrinkles, retinal folds and peripapillary hyperreflective ovoid mass-like structures associated with elevated intracranial pressure.

Background: Elevated intracranial pressure (ICP) is a serious and potentially life-threatening condition, for which clinically useful non-invasive measures have been elusive, in some cases due to their inadequate sensitivity and specificity. Our aim was to evaluate novel non-invasive ophthalmic imaging of selected pathological features seen in elevated ICP, namely peripapillary hyperreflective ovoid mass-like structures (PHOMS), peripapillary wrinkles (PPW) and retinal folds (RF) as potential biomarkers of elevated ICP. Methods: This single-center pilot study included subjects with untreated or incompletely treated high ICP. The retinas of these subjects were evaluated with averaged en-face optical coherence tomography (OCT), OCT retinal cross-sections (OCT B-scans), adaptive optics scanning light ophthalmoscopy (AOSLO), and fundus photos. Results: Seven subjects were included in the study. 6 subjects with high ICP (5 idiopathic intracranial hypertension, 1 medication induced, 30.8 ± 8.6 years, 75% female, 5 with papilledema) and 1 control (20-25 years) were included. PHOMS, PPW and RF were present in all subjects with papilledema, but neither in the high ICP subject without papilledema nor in the control subject. Averaged en-face OCT scans and AOSLO were more sensitive for PPW and RF than OCT B-scans and commercial fundus photos. Conclusion: PPW, RF and PHOMS volume have potential as non-invasive biomarkers of ICP. Novel imaging modalities may improve sensitivity. However, lack of automated image acquisition and processing limits current widespread adoption in clinical settings. Further research is needed to validate these structures as biomarkers for elevated ICP and improve clinical utility.

Quantification of Human Photoreceptor-Retinal Pigment Epithelium Macular Topography with Adaptive Optics-Optical Coherence Tomography.

Photoreceptors (PRs) and retinal pigment epithelial (RPE) cells form a functional unit called the PR-RPE complex. The PR-RPE complex plays a critical role in maintaining retinal homeostasis and function, and the quantification of its structure and topographical arrangement across the macula are important for understanding the etiology, mechanisms, and progression of many retinal diseases. However, the three-dimensional cellular morphology of the PR-RPE complex in living human eyes has not been completely described due to limitations in imaging techniques. We used the cellular resolution and depth-sectioning capabilities of a custom, high-speed Fourier domain mode-locked laser-based adaptive optics-optical coherence tomography (FDML-AO-OCT) platform to characterize human PR-RPE complex topography across the temporal macula from eleven healthy volunteers. With the aid of a deep learning algorithm, key metrics were extracted from the PR-RPE complex of averaged AO-OCT volumes including PR and RPE cell density, PR outer segment length (OSL), and PR/RPE ratio. We found a tight grouping among our cohort for PR density, with a mean (±SD) value of 53,329 (±8106) cells/mm2 at 1° decreasing to 8669 (±737) cells/mm2 at 12°. We observed a power function relationship between eccentricity and both PR density and PR/RPE ratio. We found similar variability in our RPE density measures, with a mean value of 7335 (±681) cells/mm2 at 1° decreasing to 5547 (±356) cells/mm2 at 12°, exhibiting a linear relationship with a negative slope of -123 cells/mm2 per degree. OSL monotonically decreased from 33.3 (±2.4) µm at 1° to 18.0 (±1.8) µm at 12°, following a second-order polynomial relationship. PR/RPE ratio decreased from 7.3 (±0.9) µm at 1° to 1.5 (±0.1) µm at 12°. The normative data from this investigation will help lay a foundation for future studies of retinal pathology.

The Surviving, Not Thriving, Photoreceptors in Patients with ABCA4 Stargardt Disease.

Stargardt disease (STGD1), associated with biallelic variants in the ABCA4 gene, is the most common heritable macular dystrophy and is currently untreatable. To identify potential treatment targets, we characterized surviving STGD1 photoreceptors. We used clinical data to identify macular regions with surviving STGD1 photoreceptors. We compared the hyperreflective bands in the optical coherence tomographic (OCT) images that correspond to structures in the STGD1 photoreceptor inner segments to those in controls. We used adaptive optics scanning light ophthalmoscopy (AO-SLO) to study the distribution of cones and AO-OCT to evaluate the interface of photoreceptors and retinal pigment epithelium (RPE). We found that the profile of the hyperreflective bands differed dramatically between patients with STGD1 and controls. AO-SLOs showed patches in which cone densities were similar to those in healthy retinas and others in which the cone population was sparse. In regions replete with cones, there was no debris at the photoreceptor-RPE interface. In regions with sparse cones, there was abundant debris. Our results raise the possibility that pharmaceutical means may protect surviving photoreceptors and so mitigate vision loss in patients with STGD1.

Multimodal phenotyping of foveal hypoplasia in albinism and albino-like conditions: a pediatric case series with adaptive optics insights.

Aim of the present study is to evaluate the relationship between genetic and phenotypic data in a series of patients affected by grade I and II of foveal hypoplasia with stable fixation and good visual acuity using multimodal imaging techniques. All patients underwent complete clinical and instrumental assessment including structural Optical Coherence Tomography (OCT), OCT Angiography and Adaptive Optics (AO) imaging. Central macular thickness (CMT), inner nuclear layer (INL), vessel density in superficial capillary plexus were the main variables evaluated with OCT technology. Cone density, cone spacing, cone regularity, cone dispersion and angular density were the parameters evaluated with AO. Genetic evaluation and trio exome sequencing were performed in all affected individuals. Eight patients (3 males and 5 females) with a mean age of 12.62 years (range 8-18) were enrolled. The mean best corrected visual acuity (BCVA) was 0.18 ± 0.13 logMAR, mean CMT was 291.9 ± 16.6 µm and INL was 26.2 ± 4.6 µm. The absence of a foveal avascular zone (FAZ) was documented by examination of OCT-A in seven patients in the superficial capillary plexus. However, there was a partial FAZ in the deep plexus in patients P5 and P8. Of note, all the patients presented with major retinal vessels clearly crossing the foveal center. All individuals exhibited a grade I or II of foveal hypoplasia. In 5 patients molecular analyses showed an extremely mild form of albinism caused by compound heterozygosity of a TYR pathogenic variant and the hypomorphic p.[Ser192Tyr;Arg402Gln] haplotype. One patient had Waardenburg syndrome type 2A caused by a de novo variant in MITF. Two patients had inconclusive molecular analyses. All the patients displayed abnormalities on OCT-A. Photoreceptor count did not differ from normal subjects according to the current literature, but qualitative analysis of AO imaging showed distinctive features likely related to an abnormal pigment distribution in this subset of individuals. In patients with foveal hypoplasia, genetic and multimodal imaging data, including AO findings, can help understand the physiopathology of the foveal hypoplasia phenotype. This study confirms that cone density and visual function can both be preserved despite the absence of a pit.

Multispectral label-free in vivo cellular imaging of human retinal pigment epithelium using adaptive optics fluorescence lifetime ophthalmoscopy improves feasibility for low emission analysis and increases sensitivity for detecting changes with age and eccentricity.

Significance: Adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) provides a label-free approach to observe functional and molecular changes at cellular scale in vivo. Adding multispectral capabilities improves interpretation of lifetime fluctuations due to individual fluorophores in the retinal pigment epithelium (RPE). Aim: To quantify the cellular-scale changes in autofluorescence with age and eccentricity due to variations in lipofuscin, melanin, and melanolipofuscin in RPE using multispectral AOFLIO. Approach: AOFLIO was performed on six subjects at seven eccentricities. Four imaging channels ( λ ex / λ em ) were used: 473/SSC, 473/LSC, 532/LSC, and 765/NIR. Cells were segmented and the timing signals of each pixel in a cell were combined into a single histogram, which were then used to compute the lifetime and phasor parameters. An ANOVA was performed to investigate eccentricity and spectral effects on each parameter. Results: A repeatability analysis revealed < 11.8 % change in lifetime parameters in repeat visits for 532/LSC. The 765/NIR and 532/LSC had eccentricity and age effects similar to previous reports. The 473/LSC had a change in eccentricity with mean lifetime and a phasor component. Both the 473/LSC and 473/SSC had changes in eccentricity in the short lifetime component and its relative contribution. The 473/SSC had no trend in eccentricity in phasor. The comparison across the four channels showed differences in lifetime and phasor parameters. Conclusions: Multispectral AOFLIO can provide a more comprehensive picture of changes with age and eccentricity. These results indicate that cell segmentation has the potential to allow investigations in low-photon scenarios such as in older or diseased subjects with the co-capture of an NIR channel (such as 765/NIR) with the desired spectral channel. This work represents the first multispectral, cellular-scale fluorescence lifetime comparison in vivo in the human RPE and may be a useful method for tracking diseases.