Confocal Adaptive Optics Scanning Light Ophthalmoscopy Research Articles

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

PurposeTo characterize retinal structural biomarkers for progression in adult-onset Stargardt disease from multimodal retinal imaging in-vivo maps.MethodsSeven 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.ResultsIn 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.ConclusionDark 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.

Quantifying image quality in AOSLO images of photoreceptors.

The use of "quality" to describe the usefulness of an image is ubiquitous but is often subject to domain specific constraints. Despite its continued use as an imaging modality, adaptive optics scanning light ophthalmoscopy (AOSLO) lacks a dedicated metric for quantifying the quality of an image of photoreceptors. Here, we present an approach to evaluating image quality that extracts an estimate of the signal to noise ratio. We evaluated its performance in 528 images of photoreceptors from two AOSLOs, two modalities, and healthy or diseased retinas. The algorithm was compared to expert graders' ratings of the images and previously published image quality metrics. We found no significant difference in the SNR and grades across all conditions. The SNR and the grades of the images were moderately correlated. Overall, this algorithm provides an objective measure of image quality that closely relates to expert assessments of quality in both confocal and split-detector AOSLO images of photoreceptors.

Long-term retinal imaging of a case of suspected congenital rubella infection

PurposeMany retinal disorders present with pigmentary retinopathy, most of which are progressive conditions. Here we present over nine years of follow up on a case of stable pigmentary retinopathy that is suspected to stem from a congenital rubella infection. Parafoveal cone photoreceptors were tracked through this period to gain insight into photoreceptor disruption in this pigmentary retinopathy. MethodsThe patient was examined at 8 visits spanning a total of 111 months. Examination at baseline included clinical fundus examination, full-field electroretinography (ERG), kinetic visual field assessment (Goldmann), and best corrected visual acuity; all of these except ERG were repeated at follow up visits. Imaging was performed with fundus photography, spectral-domain optical coherence tomography (SD-OCT) and confocal adaptive optics scanning light ophthalmoscopy (AOSLO). For the latter four time points AOSLO imaging also included split-detector imaging. ResultsThere were no defects in hearing or cardiac health found in this patient. There were minimal visual deficits found at baseline, with mild rod suppression on ERG; best corrected visual acuity was 20/25 OD and 20/20 OS at baseline, which was stable throughout the follow-up period. Retinal thickness as measured by OCT was within the normal range, though foveal hypoplasia was present and outer nuclear layer thickness was slightly below the normal range at all time points. Cone density was relatively stable throughout the follow-up period. A number of cones were non-reflective when observed with confocal AOSLO imaging and density was markedly lower than expected values (foveal cone density was 43,782 cones/mm2 on average). Genetic analysis revealed no causative variations explaining the phenotype. Conclusions and ImportanceThis patient appears to have a stable pigmentary retinopathy. This case is likely due to a congenital insult, rather than progressive retinal disease. This finding of stability agrees with other reports of rubella pigmentary retinopathy. Imaging with AOSLO enabled observation of two notable phenotypic features. First is the observation of dark cones, which are seen in many retinal disorders including color vision defects and degenerative retinal disease. Second, the cone density is well below what is expected – this is especially interesting as this patient has near-normal visual acuity despite this greatly decreased number of normally-waveguiding cones in the fovea.

Longitudinal Assessment of Remnant Foveal Cone Structure in a Case Series of Early Macular Telangiectasia Type 2.

PurposeTo determine the extent of remnant cone structure within early foveal ellipsoid zone (EZ) lesions in macular telangiectasia type 2 longitudinally using both confocal and split detector adaptive optics scanning light ophthalmoscopy (AOSLO).MethodsSpectral domain optical coherence tomography (SDOCT), confocal and split detector AOSLO were acquired from seven patients (10 eyes) with small (early) EZ lesions on SDOCT secondary to macular telangiectasia type 2 at baseline, 6 months, and 12 months. The presence of cone structure on AOSLO in areas of EZ loss as well as cones at 1° eccentricity, and their change over time were quantified.ResultsBy split detector AOSLO, remnant cone structure was identified within and on the borders of all foveal EZ lesions. Within the extent of these lesions, cone spacing ranged from 4.97 to 9.95 µm at baseline, 5.30 to 6.10 µm at 6 months, and 4.99 to 7.12 µm at 12 months. Four eyes with significantly smaller EZ lesions showed evidence of recovery of EZ reflectivity on SDOCT B-scans. Remnant cone structure was identified in some areas where EZ reflectivity recovered at the following time point. Eyes that showed recovery of EZ reflectivity had a continuous external limiting membrane.ConclusionsRemnant cone structure can persist within small SDOCT-defined EZ lesions, which can wax and wane in appearance over time. AOSLO can help to inform the interpretation of SDOCT imaging.Translational RelevanceThe absence of EZ in early macular telangiectasia type 2 and other retinal conditions needs careful interpretation because it does not always indicate an absence of underlying cone structure. The integrity of the external limiting membrane may better predict the presence of remnant cone structure and recovery of EZ reflectivity.

Characterization of Retinal Structure in ATF6-Associated Achromatopsia.

PurposeMutations in six genes have been associated with achromatopsia (ACHM): CNGA3, CNGB3, PDE6H, PDE6C, GNAT2, and ATF6. ATF6 is the most recent gene to be identified, though thorough phenotyping of this genetic subtype is lacking. Here, we sought to test the hypothesis that ATF6-associated ACHM is a structurally distinct form of congenital ACHM.MethodsSeven genetically confirmed subjects from five nonconsanguineous families were recruited. Foveal hypoplasia and the integrity of the ellipsoid zone (EZ) band (a.k.a., IS/OS) were graded from optical coherence tomography (OCT) images. Images of the photoreceptor mosaic were acquired using confocal and nonconfocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Parafoveal cone and rod density values were calculated and compared to published normative data as well as data from two subjects harboring CNGA3 or CNGB3 mutations who were recruited for comparative purposes. Additionally, nonconfocal dark-field AOSLO images of the retinal pigment epithelium were obtained, with quantitative analysis performed in one subject with ATF6-ACHM.ResultsFoveal hypoplasia was observed in all subjects with ATF6 mutations. Absence of the EZ band within the foveal region (grade 3) or appearance of a hyporeflective zone (grade 4) was seen in all subjects with ATF6 using OCT. There was no evidence of remnant foveal cone structure using confocal AOSLO, although sporadic cone-like structures were seen in nonconfocal split-detection AOSLO. There was a lack of cone structure in the parafovea, in direct contrast to previous reports.ConclusionsOur data demonstrate a near absence of cone structure in subjects harboring ATF6 mutations. This implicates ATF6 as having a major role in cone development and suggests that at least a subset of subjects with ATF6-ACHM have markedly fewer cellular targets for cone-directed gene therapies than do subjects with CNGA3- or CNGB3-ACHM.

A 2-Year Longitudinal Study of Normal Cone Photoreceptor Density.

PurposeDespite the potential for adaptive optics scanning light ophthalmoscopy (AOSLO) to quantify retinal disease progression at the cellular level, there remain few longitudinal studies investigating changes in cone density as a measure of disease progression. Here, we undertook a prospective, longitudinal study to investigate the variability of cone density measurements in normal subjects during a 2-year period.MethodsFourteen eyes of nine subjects with no known ocular pathology were imaged both at a baseline and a 2-year follow-up visit by using confocal AOSLO at five retinal locations. Two-year affine-registered images were created to minimize the effects of intraframe distortions. Regions of interest were cropped from baseline, 2-year manually aligned, and 2-year affine-registered images. Cones were identified (graded masked) and cone density was extracted.ResultsMean baseline cone density (cones/mm2) was 87,300, 62,200, 45,500, 28,700, and 18,200 at 190, 350, 500, 900, and 1500 μm, respectively. The mean difference (± standard deviation [SD]) in cone density from baseline to 2-year affine-registered images was 1400 (1700), 100 (1800), 300 (800), 400 (800), and 1000 (2400) cones/mm2 at the same locations. The mean difference in cone density during the 2-year period was lower for affine-registered images than manually aligned images.ConclusionsThere was no meaningful change in normal cone density during a 2-year period. Intervisit variability in cone density measurements decreased when intraframe distortions between time points were minimized. This variability must be considered when planning prospective longitudinal clinical trials using changes in cone density as an outcome measure for assessing retinal disease progression.

CELLULAR IMAGING OF THE TAPETAL-LIKE REFLEX IN CARRIERS OF RPGR-ASSOCIATED RETINOPATHY.

To examine the features of the tapetal-like reflex (TLR) in female carriers of RPGR-associated retinopathy by means of adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography. Nine molecularly confirmed RPGR carriers and three healthy controls underwent ocular examination and the following retinal imaging modalities: color photography, near-infrared reflectance, fundus autofluorescence, spectral domain optical coherence tomography, and AOSLO. After identifying TLR areas across all imaging modalities, normalized local contrast of outer retinal bands on spectral domain optical coherence tomography was calculated and AOSLO-acquired photoreceptor mosaic analysis was performed. Seven carriers had TLR areas, which colocalized with increased rod photoreceptor reflectivity on confocal AOSLO and reduced cone photoreceptor densities. Parafoveal TLR areas also exhibited reduced local contrast (i.e., increased reflectivity) of the outer retinal bands on spectral domain optical coherence tomography (inner segment ellipsoid zone and outer segment interdigitation zone). Healthy controls did not show TLR. The cellular resolution provided by AOSLO affords the characterization of the photoreceptor mosaic in RPGR carriers with a TLR. Features revealed include reduced cone density, increased cone inner segment diameter, and increased rod outer segment reflectivity.

Assessing photoreceptor structure in patients with traumatic head injury

ObjectivePrevious work using adaptive optics scanning light ophthalmoscopy (AOSLO) imaging has shown photoreceptor disruption to be a common finding in head and ocular trauma patients. Here an expanded trauma population...

The Reliability of Cone Density Measurements in the Presence of Rods.

PurposeRecent advances in adaptive optics scanning light ophthalmoscopy (AOSLO) have enabled visualization of cone inner segments through nonconfocal split-detection, in addition to rod and cone outer segments revealed by confocal reflectance. Here, we examined the interobserver reliability of cone density measurements in both AOSLO imaging modalities.MethodsFive normal subjects (nine eyes) were imaged along the horizontal and vertical meridians using a custom AOSLO with confocal and nonconfocal split-detection modalities. The resulting images were montaged using a previously described semiautomatic algorithm. Regions of interest (ROIs) were selected from the confocal montage at 190 μm, and from split-detection and confocal montages at 900 and 1800 μm from the fovea. Four observers (three experts, one naïve) manually identified cone locations in each ROI, and these locations were used to calculate bound densities. Intraclass correlation coefficients and Dice's coefficients were calculated to assess interobserver agreement.ResultsInterobserver agreement was high in cone-only images (confocal 190 μm: 0.85; split-detection 900 μm: 0.91; split-detection 1800 μm: 0.89), moderate in confocal images at 900 μm (0.68), and poor in confocal images at 1800 μm (0.24). Excluding the naïve observer data substantially increased agreement within confocal images (190 μm: 0.99; 900 μm: 0.80; 1800 μm: 0.68).ConclusionsInterobserver measurements of cone density are more reliable in rod-free retinal images. Moreover, when using manual cell identification, it is essential that observers are trained, particularly for confocal AOSLO images.Translational RelevanceThis study underscores the need for additional reliability studies in eyes containing pathology where identifying cones can be substantially more difficult.

PHOTORECEPTOR INNER SEGMENT MORPHOLOGY IN BEST VITELLIFORM MACULAR DYSTROPHY.

To characterize outer retina structure in best vitelliform macular dystrophy (BVMD) and to determine the effect of macular lesions on overlying and adjacent photoreceptors. Five individuals with BVMD were followed prospectively with spectral domain optical coherence tomography and confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). The AOSLO cone photoreceptor mosaic images were obtained within and around retinal lesions. Cone density was measured inside and outside lesions. In 2 subjects, densities were compared with published measurements acquired ∼2.5 years before. One subject was imaged 3 times over a 5-month period. The AOSLO imaging demonstrated that photoreceptor morphology within BVMD retinal lesions was highly variable depending on the disease stage, with photoreceptor structure present even in advanced disease. The AOSLO imaging was repeatable even in severe disease over short-time and long-time intervals. Photoreceptor density was normal in retinal areas immediately adjacent to lesions and stable over ∼2.5 years. Mobile disk-like structures possibly representing subretinal macrophages were also observed. Combined confocal and nonconfocal split-detector AOSLO imaging reveals substantial variability within clinical lesions in all stages of BVMD. Longitudinal cellular photoreceptor imaging could prove a powerful tool for understanding disease progression and monitoring emerging therapeutic treatment response in inherited degenerations such as BVMD.

Imaging individual neurons in the retinal ganglion cell layer of the living eye

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Assessing Photoreceptor Structure in Retinitis Pigmentosa and Usher Syndrome.

PurposeThe purpose of this study was to examine cone photoreceptor structure in retinitis pigmentosa (RP) and Usher syndrome using confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO).MethodsNineteen subjects (11 RP, 8 Usher syndrome) underwent ophthalmic and genetic testing, spectral-domain optical coherence tomography (SD-OCT), and AOSLO imaging. Split-detector images obtained in 11 subjects (7 RP, 4 Usher syndrome) were used to assess remnant cone structure in areas of altered cone reflectivity on confocal AOSLO.ResultsDespite normal interdigitation zone and ellipsoid zone appearance on OCT, foveal and parafoveal cone densities derived from confocal AOSLO images were significantly lower in Usher syndrome compared with RP. This was due in large part to an increased prevalence of non-waveguiding cones in the Usher syndrome retina. Although significantly correlated to best-corrected visual acuity and foveal sensitivity, cone density can decrease by nearly 38% before visual acuity becomes abnormal. Aberrantly waveguiding cones were noted within the transition zone of all eyes and corresponded to intact inner segment structures. These remnant cones decreased in density and increased in diameter across the transition zone and disappeared with external limiting membrane collapse.ConclusionsFoveal cone density can be decreased in RP and Usher syndrome before visible changes on OCT or a decline in visual function. Thus, AOSLO imaging may allow more sensitive monitoring of disease than current methods. However, confocal AOSLO is limited by dependence on cone waveguiding, whereas split-detector AOSLO offers unambiguous and quantifiable visualization of remnant cone inner segment structure. Confocal and split-detector thus offer complementary insights into retinal pathology.

Reliability and Repeatability of Cone Density Measurements in Patients with Congenital Achromatopsia.

Adaptive optics scanning light ophthalmoscopy (AOSLO) allows non-invasive assessment of the cone photoreceptor mosaic. Confocal AOSLO imaging of patients with achromatopsia (ACHM) reveals an altered reflectivity of the remaining cone structure, making identification of the cells more challenging than in normal retinas. Recently, a "split-detector" AOSLO imaging method was shown to enable direct visualization of cone inner segments in patients with ACHM. Several studies have demonstrated gene replacement therapy effective in restoring cone function in animal models of ACHM and human trials have on the horizon, making the ability to reliably assess cone structure increasingly important. Here we sought to examine whether absolute estimates of cone density obtained from split-detector and confocal AOSLO images differed from one another and whether the inter- and intra-observer reliability is significantly different between these modes. These findings provide an important foundation for evaluating the role of these images as tools to assess the efficacy of future gene therapy trials.

Assessing Photoreceptor Structure in Macular Hole using Split-detector Adaptive Optics Scanning Light Ophthalmoscopy

Introduction:Macular hole (MH) and vitreomacular traction (VMT) can involve disruption at the level of the photoreceptor interdigitation zone (IZ) and ellipsoid zone (EZ) with optical coherence tomography (OCT). Confocal adaptive optics scanning light ophthalmoscopy (AOSLO) has been used to examine the photoreceptor mosaic following surgical intervention in patients with MH and VMT, showing large ‘dark areas’ devoid of normal waveguiding cones. Using split-detector AOSLO, which allows visualisation of cone photoreceptor inner segments, we examined the macular cone structure in these disruptions.Methods:Seven eyes from six subjects with MH or VMT were imaged with spectral domain OCT (SD-OCT), confocal AOSLO and non-confocal split-detection AOSLO following pars plana vitrectomy (PPV) for MH or intravitreal injection with ocriplasmin for VMT.Results:Split-detector AOSLO imagery revealed remnant inner segment structure within dark areas observed with confocal AOSLO. In addition, split-detector images demonstrated that not all hyperreflective dots in confocal AOSLO images were derived from cones.Conclusion:Split-detector AOSLO provides additional information for these retinal conditions, and is likely to become an invaluable tool for assessing residual cone structure in conditions where disrupted cone structure interferes with the ability to visualise cells with confocal AOSLO.