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Abstract Image based cell-specific biomarkers will play an important role in monitoring treatment outcomes of novel therapies in patients with Stargardt (STGD1) disease and may provide information on the exact mechanism of retinal degeneration. This study reports retinal image features from conventional clinical imaging and from corresponding high-resolution imaging with a confocal adaptive optics scanning laser ophthalmoscope (AOSLO) in a heterogenous cohort of patients with Stargardt (STGD1) disease. This is a prospective observational study in which 16 participants with clinically and molecularly confirmed STGD1, and 7 healthy controls underwent clinical assessment and confocal AOSLO imaging. Clinical assessment included short-wavelength and near-infrared fundus autofluorescence, spectral-domain optical coherence tomography, and macular microperimetry. AOSLO images were acquired over a range of retinal eccentricities (0°–20°) and mapped to areas of interest from the clinical images. A regular photoreceptor mosaic was identified in areas of normal or near normal retinal structure on clinical images. Where clinical imaging indicated areas of retinal degeneration, the photoreceptor mosaic was disorganised and lacked unambiguous cones. Discrete hyper-reflective foci were identified in 9 participants with STGD1 within areas of retinal degeneration. A continuous RPE cell mosaic at the fovea was identified in one participant with an optical gap phenotype. The clinical heterogeneity observed in STGD1 is reflected in the findings on confocal AOSLO imaging.

Background: Disease-causing variants in the KCNV2 gene are associated with "cone dystrophy with supernormal rod responses," a rare autosomal recessive retinal dystrophy. There is no previous report of hypomorphic variants in the disease. Material and Methods: Medical history, genetic testing, ocular examination, high-resolution retinal imaging including adaptive optics scanning light ophthalmoscopy (AOSLO), and functional assessments. Results: A 16-year-old male with mild cone-rod dystrophy presented with reduced central vision and photophobia. Genetic testing showed two variants in KCNV2, c.614_617dupAGCG (p.207AlafsTer166) and c.854T>G (p.Met285Arg), the latter which was previously considered benign. Electrophysiological assessment revealed the pathognomic electroretinogram waveforms associated with KCNV2-retinopathy. Optical coherence tomography showed discrete focal ellipsoid zone disruption, while fundus autofluorescence was normal. Non-waveguiding cones corresponding to areas of loss of photoreceptor integrity were visible on adaptive optics scanning light ophthalmoscopy. Retinal sensitivity and fixation were relatively preserved, with a demonstrable deterioration after 14 months of follow-up. Conclusions: We provide functional and structural evidence that the variant M285R is disease-causing if associated with a loss-of-function variant. To the best of our knowledge, this is the first hypomorphic allele reported in KCNV2. [ABSTRACT FROM AUTHOR]

To compare peak cone density predicted from outer segment length measured on optical coherence tomography with direct measures of peak cone density from adaptive optics scanning light ophthalmoscopy. Data from 42 healthy participants with direct peak cone density measures and optical coherence tomography line scans available were used in this study. Longitudinal reflectivity profiles were analyzed using two methods of identifying the boundaries of the ellipsoid and interdigitation zones to estimate maximum outer segment length: peak-to-peak and the slope method. These maximum outer segment length values were then used to predict peak cone density using a previously described geometrical model. A comparison between predicted and direct peak cone density measures was then performed. The mean bias between observers for estimating maximum outer segment length across methods was less than 2 µm. Cone density predicted from the peak-to-peak method against direct cone density measures showed a mean bias of 6,812 cones/mm2 with 50% of participants displaying a 10% difference or less between predicted and direct cone density values. Cone density derived from the slope method showed a mean bias of −17,929 cones/mm2 relative to direct cone density measures, with only 41% of participants demonstrating less than a 10% difference between direct and predicted cone density values. Predicted foveal cone density derived from peak-to-peak outer segment length measurements using commercial optical coherence tomography show modest agreement with direct measures of peak cone density from adaptive optics scanning light ophthalmoscopy. The methods used here are imperfect predictors of cone density, however, further exploration of this relationship could reveal a clinically relevant marker of cone structure. [ABSTRACT FROM AUTHOR]

Background: To describe longitudinal changes in patients with non-paraneoplastic autoimmune retinopathy (npAIR) by utilizing different diagnostic modalities/tests. Methods: The index study is a retrospective longitudinal review of sixteen eyes of eight patients from a tertiary care eye hospital diagnosed with npAIR. Multiple diagnostic modalities such as wide-angle fundus photography (WAFP), WA fundus autofluorescence (WAFAF), spectral-domain optical coherence tomography (SD-OCT), Goldmann visual field (GVF) perimetry, microperimetry (MP), electrophysiologic testing, and adaptive optics scanning laser ophthalmoscopy (AOSLO) were reviewed and analyzed. Results: At the baseline visits, anomalies were detected by multimodal diagnostic tests on all patients. Subjects were followed up for a median duration of 11.5 [3.0–18.7] months. Structural changes at the baseline were detected in 14 of 16 (87.5%) eyes on WAFP and WAFAF and 13 of 16 (81.2%) eyes on SD-OCT. Eight of the ten (80%) eyes that underwent AOSLO imaging depicted structural changes. Functional changes were detected in 14 of 16 (87.5%) eyes on GVF, 15 of 16 (93.7%) eyes on MP, and 11 of 16 (68.7%) eyes on full-field electroretinogram (ff-ERG). Multifocal electroretinogram (mf-ERG) and visual evoked potential (VEP) tests were performed in 14 eyes, of which 12 (85.7%) and 14 (100%) of the eyes demonstrated functional abnormalities, respectively, at baseline. Compared to all the other structural diagnostic tools, AOSLO had a better ability to demonstrate deterioration in retinal microstructures occurring at follow-ups. Functional deterioration at follow-up was detected on GVF in 8 of 10 (80%) eyes, mf-ERG in 4 of 8 (50%) eyes, and MP in 7 of 16 (43.7%) eyes. The ff-ERG and VEP were stable in the majority of cases at follow-up. Conclusions: The utilization of multimodal imaging/tests in the diagnosing and monitoring of npAIR patients can aid in identifying anomalous changes over time. Analysis of both the anatomical and functional aspects by these devices can be supportive of detecting the changes early in such patients. AOSLO shows promise as it enables the capture of high-resolution images demonstrating quantifiable changes to retinal microstructure. [ABSTRACT FROM AUTHOR]

Background: To describe longitudinal changes in patients with non-paraneoplastic autoimmune retinopathy (npAIR) by utilizing different diagnostic modalities/tests. Methods: The index study is a retrospective longitudinal review of sixteen eyes of eight patients from a tertiary care eye hospital diagnosed with npAIR. Multiple diagnostic modalities such as wide-angle fundus photography (WAFP), WA fundus autofluorescence (WAFAF), spectral-domain optical coherence tomography (SD-OCT), Goldmann visual field (GVF) perimetry, microperimetry (MP), electrophysiologic testing, and adaptive optics scanning laser ophthalmoscopy (AOSLO) were reviewed and analyzed. Results: At the baseline visits, anomalies were detected by multimodal diagnostic tests on all patients. Subjects were followed up for a median duration of 11.5 [3.0–18.7] months. Structural changes at the baseline were detected in 14 of 16 (87.5%) eyes on WAFP and WAFAF and 13 of 16 (81.2%) eyes on SD-OCT. Eight of the ten (80%) eyes that underwent AOSLO imaging depicted structural changes. Functional changes were detected in 14 of 16 (87.5%) eyes on GVF, 15 of 16 (93.7%) eyes on MP, and 11 of 16 (68.7%) eyes on full-field electroretinogram (ff-ERG). Multifocal electroretinogram (mf-ERG) and visual evoked potential (VEP) tests were performed in 14 eyes, of which 12 (85.7%) and 14 (100%) of the eyes demonstrated functional abnormalities, respectively, at baseline. Compared to all the other structural diagnostic tools, AOSLO had a better ability to demonstrate deterioration in retinal microstructures occurring at follow-ups. Functional deterioration at follow-up was detected on GVF in 8 of 10 (80%) eyes, mf-ERG in 4 of 8 (50%) eyes, and MP in 7 of 16 (43.7%) eyes. The ff-ERG and VEP were stable in the majority of cases at follow-up. Conclusions: The utilization of multimodal imaging/tests in the diagnosing and monitoring of npAIR patients can aid in identifying anomalous changes over time. Analysis of both the anatomical and functional aspects by these devices can be supportive of detecting the changes early in such patients. AOSLO shows promise as it enables the capture of high-resolution images demonstrating quantifiable changes to retinal microstructure.

Purpose: To test the hypothesis that hyperreflective foci in central serous chorioretinopathy (CSCR) are autofluorescent and may represent macrophages that have engulfed outer retinal fluorophores from the retinal pigment epithelium (RPE) and photoreceptors. Methods: Enrolled subjects underwent spectral domain and swept-source optical coherence tomography, adaptive optics flood-illumination, and adaptive optics scanning laser ophthalmoscopy (AOSLO), including near-infrared autofluorescence (AO-IRAF). For the AO-IRAF imaging, retinal fluorophores were excited using 795 nm light and collected in an emission band from 814 to 850 nm. Results: In 2 of 3 eyes, a hyperautofluorescent signal was detected with an elliptical shape and punctate, granular aspects surrounded by a hypoautofluorescent halo. The size of these structures in the active case was measured to be 17 ± 4 μm in diameter, with at least 45 individual hyperautofluorescent foci identified from the AO-IRAF montage in the active stage of patient 2. In the asymptomatic case there were fewer structures visible (∼10) and their size was smaller (11 ± 4 μm). These hyper-AF foci were colocalized with hyperreflective foci on OCT and visible in simultaneously acquired confocal AOSLO images in active stage. The hyperautofluorescent foci in the patient with active CSCR disappeared coincident with clinical resolution. Conclusion and importance: We show here the first AO-IRAF images from patients with CSCR, demonstrating hyper-autofluorescent punctate foci, colocalized with hyper-reflective foci on confocal AOSLO images and in OCT. The autofluorescence of these foci may be driven by the accumulation of photoreceptor and RPE fluorophores within macrophages during the active stage of the disease.

Purpose: To describe the reflectance adaptive optics scanning laser ophthalmoscopy (AOSLO) findings in different stages of Vogt-Koyanagi-Harada (VKH) disease and correlate them to visual gain post treatment. Confocal (cAOSLO) and non-confocal split-detector AOSLO (sdAOSLO) were used to assess longitudinally the status of the photoreceptors in a patient with VKH managed on corticosteroid and immunomodulatory therapy. Observation: A 32-year-old Japanese American female presented with a 2-week history of blurred vision in both eyes (OU) and worsening headache previously diagnosed as a case of VKH and treated with high dose oral prednisone. At the time of presentation, though vision was improving, and frank serous retinal detachments were absent, spectral domain optical coherence tomography (SD-OCT) showed presence of residual subretinal fluid with disruption of the photoreceptor inner segments and outer segments (IS/OS) involving OU. The photoreceptor mosaic at the foveal center appeared very sparse with large areas devoid of visible photoreceptors on cAOSLO, in agreement with the SD-OCT data. sdAOSLO imaging over the same location shows a higher number of contiguous photoreceptors. After imaging, the patient was started on mycophenolate mofetil as steroid-sparing long-term therapy. Three months later, visual acuity improved to 20/20 OU, and SD-OCT showed almost complete resolution of subretinal fluid with significant improvement of the IS/OS SD-OCT signal, OU. cAOSLO imaging revealed a contiguous photoreceptor mosaic without gaps and of normal appearance. Conclusions and Importance: VKH patients may demonstrate transient photoreceptor abnormalities on SD-OCT and cAOSLO imaging. sdAOSLO imaging revealed intact photoreceptor segments in areas that appeared as voids on cAOSLO, which later showed structural recovery on SD-OCT and cAOSLO. Therefore, sdAOSLO may predict potential for improvement in patients wherein there appears to be photoreceptor loss in cAOSLO and/or SD-OCT.

High resolution retinal imaging paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of activity dependent calcium changes in retinal ganglion cells (RGCs) at single cell resolution in the living eye. The inner limiting membrane (ILM) is a barrier for viral vectors, restricting transduction to a ring of RGCs serving the fovea in both humans and non-human primates (NHP). We evaluate peeling the ILM prior to intravitreal injection as a strategy to expand calcium imaging beyond the fovea in the NHP eye in vivo. Five Macaca fascicularis eyes (age 3-10y; n=3 individuals; 2M, 1F) underwent vitrectomy and 5 to 6-disc diameter ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. Calcium responses from RGCs were recorded using a fluorescence adaptive optics scanning laser ophthalmoscope. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in area of expression relative to a control eye. Calcium recordings were obtained up to 11 degrees from the foveal center. RGC responses were comparable to the fellow control eye and showed no significant decrease over the 6 months post ILM peel, suggesting that RGC function was not compromised by the surgical procedure. In addition, we demonstrate that activity can be recorded directly from the retinal nerve fiber layer. This approach will be valuable for a range of applications in visual neuroscience including pre-clinical evaluation of retinal function, detecting vision loss, and assessing the impact of therapeutic interventions., Competing Interests: The authors declare no competing financial interests.

Purpose: Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging. Methods: One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL). Results: Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects. Conclusions: This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process.

Purpose: To determine the axial location of Gunn's dots in the retina. Methods: Adaptive 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. Results: All 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. Conclusions: Based 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.

Purpose: To present ophthalmic imaging findings in the case of a 40-year-old male with sustained visual loss after a single episode of acute central serous retinopathy (CSR). Observations: A male subject presented with visual acuity decline to 20/50 OS and was diagnosed with acute CSR. The initial pigment epithelial detachment and subretinal fluid resolved within 6 weeks, but visual acuity remained impaired. Using directional optical coherence tomography (D-OCT) and confocal and split-detector adaptive optics scanning light ophthalmoscopy (AOSLO), we imaged pathologic alterations in the photoreceptor mosaic of the affected eye. A foveal region of intermittent missing cones, a temporal parafoveal region of confluent missing cones, and a nasal parafoveal region of misdirected cones were observed. Conclusions and Importance: Pathologic alterations in photoreceptor microanatomy underlie residual visual acuity deficits in this case of acute CSR. Observations of missing cones correlated well across all imaging modalities in the fovea and the temporal parafoveal region of missing cones. However, in the nasal parafovea where cones were present but misdirected, D-OCT and AOSLO may be able to identify and image photoreceptors with greater fidelity as compared to non-directional SDOCT (spectral domain OCT). D-OCT may thus have a clinical role in rapidly assessing photoreceptor mosaic integrity in pathology.

David Merino, Pablo Loza-Alvarez The Institute of Photonic Sciences (ICFO), The Barcelona Institute of Science and Technology, Castelldefels, Barcelona, Spain Abstract: Adaptive optics (AO) retinal imaging has become very popular in the past few years, especially within the ophthalmic research community. Several different retinal techniques, such as fundus imaging cameras or optical coherence tomography systems, have been coupled with AO in order to produce impressive images showing individual cell mosaics over different layers of the in vivo human retina. The combination of AO with scanning laser ophthalmoscopy has been extensively used to generate impressive images of the human retina with unprecedented resolution, showing individual photoreceptor cells, retinal pigment epithelium cells, as well as microscopic capillary vessels, or the nerve fiber layer. Over the past few years, the technique has evolved to develop several different applications not only in the clinic but also in different animal models, thanks to technological developments in the field. These developments have specific applications to different fields of investigation, which are not limited to the study of retinal diseases but also to the understanding of the retinal function and vision science. This review is an attempt to summarize these developments in an understandable and brief manner in order to guide the reader into the possibilities that AO scanning laser ophthalmoscopy offers, as well as its limitations, which should be taken into account when planning on using it. Keywords: high-resolution, in vivo retinal imaging, AOSLO

Purpose: The purpose of this study was to develop an algorithm based on region-based segmentation for automated calculations of human cone photoreceptor density of en face images obtained by an adaptive optics scanning laser ophthalmoscope (AOSLO). Subjects and methods: Cone mosaics of 15 eyes of 15 healthy subjects were photographed by a custom-built AOSLO. The cone density was calculated at 0.5, 1.0, and 1.5 mm temporal from the fovea using a region-based segmentation method (RSM) developed in our laboratory. The cone density was also determined by a manual identification method (MIM) and a conventional spatial filtering method (SFM). The cone densities of three eyes of three patients with retinal degeneration were calculated by the three methods and compared to the results from normal eyes. Results: The cone densities in healthy retinas determined by the RSM at 0.5, 1.0, and 1.5 mm temporal from the fovea were 28,436, 21,233, and 13,620 cells/mm2, respectively. These densities were in good agreement with a histological study and with in vivo AOSLO studies. The cone densities determined by RSM were different from those determined by MIM with a difference of 5% in healthy eyes. In eyes with retinal degeneration, with the appropriate threshold-level settings or spatial frequency bandwidth, the cone density measured by MIM was significantly closer to that measured by RSM than by SFM. Conclusion: These results suggest that our method is more stable than conventional methods in cases of non-periodical photoreceptor structures such as the affected retinal area. Our method can be used in the longitudinal follow-up of retinal degenerative diseases and to determine the effect of therapy. [ABSTRACT FROM AUTHOR]

Purpose: To report the results of an acetazolamide (Diamox) treatment regimen in a genetically confirmed case of X-linked juvenile retinoschisis (XLRS). Methods: A patient with XLRS was prescribed acetazolamide (Diamox) at a dose of 500 mg/day, then discontinued the treatment due to non-compliance for 4 days, and finally resumed the course of treatment. Best-corrected visual acuity, retinal structure, and function were monitored with autofluorescence, spectral domain-optical coherence tomography (SD-OCT), adaptive optics scanning laser ophthalmoscopy (AOSLO), and full-field electroretinogram (ERG). Full-field ERG was performed using DTL recording electrodes and Ganzfeld stimulation according to ISCEV standards. Results: Serial monitoring of the cysts by SD-OCT revealed a strong association between the effects of acetazolamide administration and the size of the schisis. A reduction in foveal cyst size was significant in as rapid as 6 days after acetazolamide initiation. AOSLO data revealed that the resolution of cone cell images improves as the foveal schisis decreases in size. Conclusions: Efficacy of acetazolamide in patients with XLRS can be apparent in as rapid as a week of therapy. AOSLO can be a good method to evaluate the cone cells after acetazolamide treatment in the early stages of XLRS. [ABSTRACT FROM AUTHOR]

Purpose: To test the hypothesis that hyperreflective foci in central serous chorioretinopathy (CSCR) are autofluorescent and may represent macrophages that have engulfed outer retinal fluorophores from the retinal pigment epithelium (RPE) and photoreceptors., Methods: Enrolled subjects underwent spectral domain and swept-source optical coherence tomography, adaptive optics flood-illumination, and adaptive optics scanning laser ophthalmoscopy (AOSLO), including near-infrared autofluorescence (AO-IRAF). For the AO-IRAF imaging, retinal fluorophores were excited using 795 nm light and collected in an emission band from 814 to 850 nm., Results: In 2 of 3 eyes, a hyperautofluorescent signal was detected with an elliptical shape and punctate, granular aspects surrounded by a hypoautofluorescent halo. The size of these structures in the active case was measured to be 17 ± 4 μm in diameter, with at least 45 individual hyperautofluorescent foci identified from the AO-IRAF montage in the active stage of patient 2. In the asymptomatic case there were fewer structures visible (∼10) and their size was smaller (11 ± 4 μm). These hyper-AF foci were colocalized with hyperreflective foci on OCT and visible in simultaneously acquired confocal AOSLO images in active stage. The hyperautofluorescent foci in the patient with active CSCR disappeared coincident with clinical resolution., Conclusion and Importance: We show here the first AO-IRAF images from patients with CSCR, demonstrating hyper-autofluorescent punctate foci, colocalized with hyper-reflective foci on confocal AOSLO images and in OCT. The autofluorescence of these foci may be driven by the accumulation of photoreceptor and RPE fluorophores within macrophages during the active stage of the disease., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

In vivo images of human cone photoreceptors have been shown to vary in their reflectance both spatially and temporally. While it is generally accepted that the unique anatomy and physiology of the photoreceptors themselves drives this behavior, the exact mechanisms have not been fully elucidated as most studies on these phenomena have been limited to the human retina. Unlike humans, animal models offer the ability to experimentally manipulate the retina and perform direct in vivo and ex vivo comparisons. The thirteen-lined ground squirrel and northern tree shrew are two emerging animal models being used in vision research. Both models feature cone-dominant retinas, overcoming a key limitation of traditional rodent models. Additionally, each possesses unique but well-documented anatomical differences in cone structure compared to human cones, which can be leveraged to further constrain theoretical models of light propagation within photoreceptors. Here we sought to characterize the spatial and temporal reflectance behavior of cones in these species. Adaptive optics scanning light ophthalmoscopy (AOSLO) was used to non-invasively image the photoreceptors of both species at 5 to 10 min intervals over the span of 18 to 25 min. The reflectance of individual cone photoreceptors was measured over time, and images at individual time points were used to assess the variability of cone reflectance across the cone mosaic. Variability in spatial and temporal photoreceptor reflectance was observed in both species, with similar behavior to that seen in human AOSLO images. Despite the unique cone structure in these animals, these data suggest a common origin of photoreceptor reflectance behavior across species. Such data may help constrain models of the cellular origins of photoreceptor reflectance signals. These animal models provide an experimental platform to further explore the morphological origins of light capture and propagation.

Purpose: Lafora disease is a genetic neurodegenerative metabolic disorder caused by insoluble polyglucosan aggregate accumulation throughout the central nervous system and body. The retina is an accessible neural tissue, which may offer alternative methods to assess neurological diseases quickly and noninvasively. In this way, noninvasive imaging may provide a means to characterize neurodegenerative disease, which enables earlier identification and diagnosis of disease and the ability to monitor disease progression. In this study, we sought to characterize the retina of individuals with Lafora disease using non-invasive retinal imaging., Methods: One eye of three individuals with genetically confirmed Lafora disease were imaged with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO). When possible, OCT volume and line scans were acquired to assess total retinal thickness, ganglion cell-inner plexiform layer thickness, and outer nuclear layer + Henle fiber layer thickness. OCT angiography (OCTA) scans were acquired in one subject at the macula and optic nerve head (ONH). AOSLO was used to characterize the photoreceptor mosaic and examine the retinal nerve fiber layer (RNFL)., Results: Two subjects with previous seizure activity demonstrated reduced retinal thickness, while one subject with no apparent symptoms had normal retinal thickness. All other clinical measures, as well as parafoveal cone density, were within normal range. Nummular reflectivity at the level of the RNFL was observed using AOSLO in the macula and near the ONH in all three subjects., Conclusions: This multimodal retinal imaging approach allowed us to observe a number of retinal structural features in all three individuals. Most notably, AOSLO revealed nummular reflectivity within the inner retina of each subject. This phenotype has not been reported previously and may represent a characteristic change produced by the neurodegenerative process., Competing Interests: Mrs. Linderman is a consultant for OptoVue. Dr. Rosen has personal financial interest in Opticology and Guardion, and is a consultant for OptoVue, Boehringer-Ingelheim, Astellas, Genentech-Roche, NanoRetina, OD-OS, Regeneron, and Bayer. Dr. Vincent is a consultant for Adverum Biotechnologies Inc. Dr. Carroll receives financial support from AGTC and OptoVue, is a consultant for MeiraGTX, and has personal financial interest in Translational Imaging Innovations. Dr. Minassian is chief medical advisor to Taysha Gene Therapies. The following authors have no financial disclosures: H.H., J.A.C., E.N.W., R.M., P.S., T.Y.P., and E.J.P., (© 2021 The Authors.)

Purpose: To determine the axial location of Gunn's dots in the retina., Methods: Adaptive 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., Results: All 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 μm 3 , 220.2 ± 105.5 μm 2 , and 14.3 ± 3.1 μm, the latter within the range as previously published reports., Conclusions: Based 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., Competing Interests: Dr. Carroll received non-financial support from Optovue, Inc, grants from AGTC, personal fees and non-financial support from Meira GTx, and personal fees from Translational Imaging Innovations outside the scope of the submitted work. Dr. Saeedi received personal fees and non-financial support from Heidelberg Engineering, and a grant from Vasoptic Medical Inc. outside the scope of the submitted work.

Purpose: The purpose of this study was to develop an algorithm based on region-based segmentation for automated calculations of human cone photoreceptor density of en face images obtained by an adaptive optics scanning laser ophthalmoscope (AOSLO)., Subjects and Methods: Cone mosaics of 15 eyes of 15 healthy subjects were photographed by a custom-built AOSLO. The cone density was calculated at 0.5, 1.0, and 1.5 mm temporal from the fovea using a region-based segmentation method (RSM) developed in our laboratory. The cone density was also determined by a manual identification method (MIM) and a conventional spatial filtering method (SFM). The cone densities of three eyes of three patients with retinal degeneration were calculated by the three methods and compared to the results from normal eyes., Results: The cone densities in healthy retinas determined by the RSM at 0.5, 1.0, and 1.5 mm temporal from the fovea were 28,436, 21,233, and 13,620 cells/mm 2 , respectively. These densities were in good agreement with a histological study and with in vivo AOSLO studies. The cone densities determined by RSM were different from those determined by MIM with a difference of 5% in healthy eyes. In eyes with retinal degeneration, with the appropriate threshold-level settings or spatial frequency bandwidth, the cone density measured by MIM was significantly closer to that measured by RSM than by SFM., Conclusion: These results suggest that our method is more stable than conventional methods in cases of non-periodical photoreceptor structures such as the affected retinal area. Our method can be used in the longitudinal follow-up of retinal degenerative diseases and to determine the effect of therapy., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Purpose: To evaluate visual function and disease progression in the retinal structural abnormalities of three patients from two unrelated families with macular telangiectasia (MacTel) type 2., Methods: Adaptive optics scanning laser ophthalmoscopy (AOSLO) and AOSLO microperimetry (AOMP) were used to evaluate the structure and function of macular cones in three eyes with MacTel type 2. Cone spacing was estimated using histogram analysis of intercone distances, and registered spectral-domain optical coherence tomography (SD-OCT) scans were used to evaluate retinal anatomy. AOMP was used to assess visual sensitivity in and around areas of apparent cone loss., Results: Although overall lesion surface area increased, some initially affected regions subsequently showed clear, contiguous, and normally spaced cone mosaics with recovered photoreceptor inner/outer segment (IS/OS) reflectivity (two of two eyes). The AOMP test sites fell within three categories: normal-appearing cones (N), dimly reflecting cones (D), and RPE cell mosaics (R). At N sites, AOMP threshold values (arbitrary units [au]) increased with increasing eccentricity (slope = 0.054 au/degree, r(2) = 0.77). The N thresholds ranged from 0.04 to 0.27 au, D thresholds from 0.04 to 0.33 au, and R thresholds from 0.14 to 1.00 au. There was measurable visual sensitivity everywhere except areas without intact external limiting membrane (ELM) and with diffuse scattering in the IS/OS and posterior tips of the outer segments (PTOS) regions on OCT., Conclusions: Visual sensitivity and recovery of cone visibility in areas of apparent focal cone loss suggests that MacTel type 2 lesions with a preserved ELM may contain functioning cones with abnormal scattering and/or waveguiding characteristics. (ClinicalTrials.gov number, NCT00254605.)., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: An often overlooked prerequisite to cone photoreceptor gene therapy development is residual photoreceptor structure that can be rescued. While advances in adaptive optics (AO) retinal imaging have recently enabled direct visualization of individual cone and rod photoreceptors in the living human retina, these techniques largely detect strongly directionally-backscattered (waveguided) light from normal intact photoreceptors. This represents a major limitation in using existing AO imaging to quantify structure of remnant cones in degenerating retina., Methods: Photoreceptor inner segment structure was assessed with a novel AO scanning light ophthalmoscopy (AOSLO) differential phase technique, that we termed nonconfocal split-detector, in two healthy subjects and four subjects with achromatopsia. Ex vivo preparations of five healthy donor eyes were analyzed for comparison of inner segment diameter to that measured in vivo with split-detector AOSLO., Results: Nonconfocal split-detector AOSLO reveals the photoreceptor inner segment with or without the presence of a waveguiding outer segment. The diameter of inner segments measured in vivo is in good agreement with histology. A substantial number of foveal and parafoveal cone photoreceptors with apparently intact inner segments were identified in patients with the inherited disease achromatopsia., Conclusions: The application of nonconfocal split-detector to emerging human gene therapy trials will improve the potential of therapeutic success, by identifying patients with sufficient retained photoreceptor structure to benefit the most from intervention. Additionally, split-detector imaging may be useful for studies of other retinal degenerations such as AMD, retinitis pigmentosa, and choroideremia where the outer segment is lost before the remainder of the photoreceptor cell., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Microaneurysms (MAs) are considered a hallmark of retinal vascular disease, yet what little is known about them is mostly based upon histology, not clinical observation. Here, we use the recently developed adaptive optics scanning light ophthalmoscope (AOSLO) fluorescein angiography (FA) to image human MAs in vivo and to expand on previously described MA morphologic classification schemes., Methods: Patients with vascular retinopathies (diabetic, hypertensive, and branch and central retinal vein occlusion) were imaged with reflectance AOSLO and AOSLO FA. Ninety-three MAs, from 14 eyes, were imaged and classified according to appearance into six morphologic groups: focal bulge, saccular, fusiform, mixed, pedunculated, and irregular. The MA perimeter, area, and feret maximum and minimum were correlated to morphology and retinal pathology. Select MAs were imaged longitudinally in two eyes., Results: Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging revealed microscopic features of MAs not appreciated on conventional images. Saccular MAs were most prevalent (47%). No association was found between the type of retinal pathology and MA morphology (P = 0.44). Pedunculated and irregular MAs were among the largest MAs with average areas of 4188 and 4116 μm(2), respectively. Focal hypofluorescent regions were noted in 30% of MAs and were more likely to be associated with larger MAs (3086 vs. 1448 μm(2), P = 0.0001)., Conclusions: Retinal MAs can be classified in vivo into six different morphologic types, according to the geometry of their two-dimensional (2D) en face view. Adaptive optics scanning light ophthalmoscope fluorescein angiography imaging of MAs offers the possibility of studying microvascular change on a histologic scale, which may help our understanding of disease progression and treatment response.