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1.  In Vivo Imaging of Human Cone Photoreceptor Inner Segments 
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.
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.
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.
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.
A new ophthalmic imaging technique that reveals the photoreceptor inner segment is presented and validated. The presence of retained cone photoreceptors is demonstrated in the inherited disease achromatopsia, which has significant impact for upcoming gene therapy trials.
PMCID: PMC4095721  PMID: 24906859
AOSLO; photoreceptor; gene therapy
2.  Relationship Between Foveal Cone Specialization and Pit Morphology in Albinism 
Albinism is associated with disrupted foveal development, though intersubject variability is becoming appreciated. We sought to quantify this variability, and examine the relationship between foveal cone specialization and pit morphology in patients with a clinical diagnosis of albinism.
We recruited 32 subjects with a clinical diagnosis of albinism. DNA was obtained from 25 subjects, and known albinism genes were analyzed for mutations. Relative inner and outer segment (IS and OS) lengthening (fovea-to-perifovea ratio) was determined from manually segmented spectral domain-optical coherence tomography (SD-OCT) B-scans. Foveal pit morphology was quantified for eight subjects from macular SD-OCT volumes. Ten subjects underwent imaging with adaptive optics scanning light ophthalmoscopy (AOSLO), and cone density was measured.
We found mutations in 22 of 25 subjects, including five novel mutations. All subjects lacked complete excavation of inner retinal layers at the fovea, though four subjects had foveal pits with normal diameter and/or volume. Peak cone density and OS lengthening were variable and overlapped with that observed in normal controls. A fifth hyper-reflective band was observed in the outer retina on SD-OCT in the majority of the subjects with albinism.
Foveal cone specialization and pit morphology vary greatly in albinism. Normal cone packing was observed in the absence of a foveal pit, suggesting a pit is not required for packing to occur. The degree to which retinal anatomy correlates with genotype or visual function remains unclear, and future examination of larger patient groups will provide important insight on this issue.
Foveal specialization (foveal cone packing, outer segment elongation, pit morphology) in albinism is highly variable and can overlap with normal foveal structure, contrary to the typical clinical picture of albinism.
PMCID: PMC4098060  PMID: 24845642
albinism; adaptive optics; foveal morphology; foveal development
3.  Retinal Structure and Function in Achromatopsia: Implications for Gene Therapy 
Ophthalmology  2013;121(1):234-245.
To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy.
Cross-sectional study.
Forty subjects with ACHM.
All subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (i) continuous inner segment ellipsoid (ISe), (ii) ISe disruption, (iii) ISe absence, (iv) presence of a hyporeflective zone (HRZ), and (v) outer retinal atrophy including retinal pigment epithelial (RPE) loss. Foveal and outer nuclear layer (ONL) thickness was measured, and presence of hypoplasia determined.
Main Outcome Measures
Photoreceptor appearance on SD-OCT imaging; foveal and ONL thickness; presence of foveal hypoplasia; retinal sensitivity and fixation stability; and association of these parameters with age and genotype.
Forty subjects with mean age of 24.9 years (range 6 to 52) were included. Disease-causing variants were found in CNGA3 (n=18), CNGB3 (n=15), GNAT2 (n=4), and PDE6C (n=1). No variants were found in 2 individuals. 22.5% of subjects had a continuous ISe layer at the fovea; 27.5% had ISe disruption; 20% had an absent ISe layer; 22.5% had a HRZ; and 7.5% had outer retinal atrophy. No significant differences in age (p=0.77), mean retinal sensitivity (p=0.21) or fixation stability (p=0.34) across the 5 SD-OCT categories were evident. No significant correlation was found between age and foveal thickness (p=0.84), or between age and foveal ONL thickness (p=0.12).
The lack of clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring their impact.
PMCID: PMC3895408  PMID: 24148654
4.  Microscopic Inner Retinal Hyper-Reflective Phenotypes in Retinal and Neurologic Disease 
We surveyed inner retinal microscopic features in retinal and neurologic disease using a reflectance confocal adaptive optics scanning light ophthalmoscope (AOSLO).
Inner retinal images from 101 subjects affected by one of 38 retinal or neurologic conditions and 11 subjects with no known eye disease were examined for the presence of hyper-reflective features other than vasculature, retinal nerve fiber layer, and foveal pit reflex. The hyper-reflective features in the AOSLO images were grouped based on size, location, and subjective texture. Clinical imaging, including optical coherence tomography (OCT), scanning laser ophthalmoscopy, and fundus photography was analyzed for comparison.
Seven categories of hyper-reflective inner retinal structures were identified, namely punctate reflectivity, nummular (disc-shaped) reflectivity, granular membrane, waxy membrane, vessel-associated membrane, microcysts, and striate reflectivity. Punctate and nummular reflectivity also was found commonly in normal volunteers, but the features in the remaining five categories were found only in subjects with retinal or neurologic disease. Some of the features were found to change substantially between follow up imaging months apart.
Confocal reflectance AOSLO imaging revealed a diverse spectrum of normal and pathologic hyper-reflective inner and epiretinal features, some of which were previously unreported. Notably, these features were not disease-specific, suggesting that they might correspond to common mechanisms of degeneration or repair in pathologic states. Although prospective studies with larger and better characterized populations, along with imaging of more extensive retinal areas are needed, the hyper-reflective structures reported here could be used as disease biomarkers, provided their specificity is studied further.
The human inner retina was examined with confocal adaptive optics scanning light ophthalmoscopy. Seven non–disease-specific categories of hyper-reflective structures were identified, suggesting common mechanisms of degeneration or repair in pathologic states.
PMCID: PMC4078949  PMID: 24894394
adaptive optics; inner retina; ophthalmoscopy; neuro-ophthalmology; retinal disease
5.  Human Cone Visual Pigment Deletions Spare Sufficient Photoreceptors to Warrant Gene Therapy 
Human Gene Therapy  2013;24(12):993-1006.
Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5–58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.
PMCID: PMC3868405  PMID: 24067079
6.  Selective Cone Photoreceptor Injury in Acute Macular Neuroretinopathy 
Retina (Philadelphia, Pa.)  2013;33(8):1650-1658.
To evaluate retinal structural and functional abnormalities in a patient with Acute Macular Neuroretinopathy (AMN).
An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Microperimetry was used to assess function across the macula.
Microperimetry showed reduced function of localized areas within retinal lesions corresponding to subjective scotomas. SD-OCT imaging revealed attenuation of two outer retinal bands typically thought to reflect photoreceptor structure. AOSLO images of the photoreceptor mosaic revealed a heterogeneous presentation within these lesions. There were areas containing non-waveguiding cones and other areas of decreased cone density where the remaining rods had expanded to fill in the vacant space. Within these lesions, cone densities were shown to be significantly lower than eccentricity matched areas of normal retina, as well as accepted histological measurements. A 6-month follow up revealed no change in rod or cone structure.
Imaging of AMN using an AOSLO shows a preferential disruption of cone photoreceptor structure within the region of decreased retinal sensitivity (as measured by microperimetry). AO-based imaging tools provide a noninvasive way to assess photoreceptor structure at a level of detail that is not resolved by use of conventional SD-OCT or other clinical measures.
PMCID: PMC4065781  PMID: 23615345
Acute Macular Neuroretinopathy; Adaptive Optics; Cones; Macula; Optical Coherence Tomography; Photoreceptors; Retinal Imaging
7.  Classification of Human Retinal Microaneurysms Using Adaptive Optics Scanning Light Ophthalmoscope Fluorescein Angiography 
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.
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.
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 μm2, respectively. Focal hypofluorescent regions were noted in 30% of MAs and were more likely to be associated with larger MAs (3086 vs. 1448 μm2, P = 0.0001).
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.
With adaptive optics scanning light ophthalmoscope fluorescein angiography (AOSLO FA), microaneurysms (MA) were classified into six distinct morphologies and measured quantitatively with a high degree of interuser reproducibility. Microaneurysm stability, growth, and regression were noted in serial exams.
PMCID: PMC3943418  PMID: 24425852
microaneurysm; AOSLO; fluorescein angiography
8.  Outer Retinal Structure in Best Vitelliform Macular Dystrophy 
JAMA ophthalmology  2013;131(9):1207-1215.
To characterize outer retinal structure in Best Vitelliform Macular Dystrophy (BVMD), using spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO).
Four symptomatic members of a family with BVMD with known BEST1 gene mutation were recruited. Thickness of two outer retinal layers corresponding to photoreceptor inner and outer segments were measured using SD-OCT. Photoreceptor mosaic AOSLO images within and around visible lesions were obtained, and cone density was assessed in two subjects.
Each subject was at a different stage of BVMD, with photoreceptor disruption evident by AOSLO at all stages. When comparing SD-OCT and AOSLO images from the same location, AOSLO images allowed for direct assessment of photoreceptor structure. A variable degree of retained photoreceptors was seen within all lesions. The photoreceptor mosaic immediately adjacent to visible lesions appeared contiguous and was of normal density. Fine hyperreflective structures were visualized by AOSLO, and their anatomical orientation and size are consistent with Henle fibers.
AOSLO findings indicate substantial photoreceptor structure persists within active lesions, accounting for good visual acuity in these patients. Despite previous reports of diffuse photoreceptor outer segment abnormalities in BVMD, our data reveal normal photoreceptor structure in areas adjacent to clinical lesions.
Clinical Relevance
This study demonstrates the utility of AOSLO for understanding the spectrum of cellular changes that occur in inherited degenerations such as BVMD. Photoreceptors are often significantly affected at various stages of inherited degenerations, and these changes may not be readily apparent with current clinical imaging instrumentation.
PMCID: PMC3968428  PMID: 23765342
9.  Non-common path aberration correction in an adaptive optics scanning ophthalmoscope 
Biomedical Optics Express  2014;5(9):3059-3073.
The correction of non-common path aberrations (NCPAs) between the imaging and wavefront sensing channel in a confocal scanning adaptive optics ophthalmoscope is demonstrated. NCPA correction is achieved by maximizing an image sharpness metric while the confocal detection aperture is temporarily removed, effectively minimizing the monochromatic aberrations in the illumination path of the imaging channel. Comparison of NCPA estimated using zonal and modal orthogonal wavefront corrector bases provided wavefronts that differ by ~λ/20 in root-mean-squared (~λ/30 standard deviation). Sequential insertion of a cylindrical lens in the illumination and light collection paths of the imaging channel was used to compare image resolution after changing the wavefront correction to maximize image sharpness and intensity metrics. Finally, the NCPA correction was incorporated into the closed-loop adaptive optics control by biasing the wavefront sensor signals without reducing its bandwidth.
PMCID: PMC4230870  PMID: 25401020
(110.1080) Active or adaptive optics; (170.4460) Ophthalmic optics and devices
10.  Automatic detection of modal spacing (Yellott’s ring) in AOSLO images 
An impediment for the clinical utilisation of ophthalmic adaptive optics imaging systems is the automated assessment of photoreceptor mosaic integrity. Here we propose a fully automated algorithm for estimating photoreceptor density based on the radius of Yellott’s ring.
The discrete Fourier transform (DFT) was used to obtain the power spectrum for a series of images of the human photoreceptor mosaic. Cell spacing is estimated by least-square fitting an annular pattern with a Gaussian cross section to the power spectrum; the radius of the resulting annulus provides an estimate of the modal spacing of the photoreceptors in the retinal image. The intrasession repeatability of the cone density estimates from the algorithm was evaluated, and the accuracy of the algorithm was validated against direct count estimates from a previous study. Accuracy in the presence of multiple cell types and disruptions in the mosaic was examined using images from 4 patients with retinal pathology and perifoveal images from 2 subjects with normal vision.
Intrasession repeatability of the power spectrum method was comparable to a fully automated direct counting algorithm, but worse than that for the manually adjusted direct count values. In images of the normal parafoveal cone mosaic, we find good agreement between the power-spectrum derived density and that from the direct counting algorithm. In diseased eyes, the power spectrum method is insensitive to photoreceptor loss, with cone density estimates overestimating the density determined with direct counting. The automated power spectrum method also produced unreliable estimates of rod and cone density in perifoveal images of the photoreceptor mosaic, though manual correction of the initial algorithm output results in density estimates in better agreement with direct count values.
We developed and validated an automated algorithm based on the power spectrum for extracting estimates of cone spacing, from which estimates of density can be derived. This approach may be used to estimate cone density in images where not every single cone is visible, though caution is needed, as this robustness becomes a weakness when dealing with images from patients with some retinal diseases. This study represents an important first step in carefully assessing the relative utility of metrics for analysing the photoreceptor mosaic, and similar analyses of other metrics/algorithms are needed.
PMCID: PMC3690144  PMID: 23668233
retinal imaging; adaptive optics; photoreceptor; repeatability
11.  Adaptive Optics Retinal Imaging – Clinical Opportunities and Challenges 
Current eye research  2013;38(7):709-721.
The array of therapeutic options available to clinicians for treating retinal disease is expanding. With these advances comes the need for better understanding of the etiology of these diseases on a cellular level as well as improved non-invasive tools for identifying the best candidates for given therapies and monitoring the efficacy of those therapies. While spectral domain optical coherence tomography (SD-OCT) offers a widely available tool for clinicians to assay the living retina, it suffers from poor lateral resolution due to the eye’s monochromatic aberrations. Adaptive optics (AO) is a technique to compensate for the eye’s aberrations and provide nearly diffraction-limited resolution. The result is the ability to visualize the living retina with cellular resolution. While AO is unquestionably a powerful research tool, many clinicians remain undecided on the clinical potential of AO imaging – putting many at a crossroads with respect to adoption of this technology. This review will briefly summarize the current state of AO retinal imaging, discuss current as well as future clinical applications of AO retinal imaging, and finally provide some discussion of research needs to facilitate more widespread clinical use.
PMCID: PMC4031042  PMID: 23621343
retinal imaging; adaptive optics; retinal degeneration; photoreceptor
12.  Comparison of adaptive optics scanning light ophthalmoscopic fluorescein angiography and offset pinhole imaging 
Biomedical Optics Express  2014;5(4):1173-1189.
Recent advances to the adaptive optics scanning light ophthalmoscope (AOSLO) have enabled finer in vivo assessment of the human retinal microvasculature. AOSLO confocal reflectance imaging has been coupled with oral fluorescein angiography (FA), enabling simultaneous acquisition of structural and perfusion images. AOSLO offset pinhole (OP) imaging combined with motion contrast post-processing techniques, are able to create a similar set of structural and perfusion images without the use of exogenous contrast agent. In this study, we evaluate the similarities and differences of the structural and perfusion images obtained by either method, in healthy control subjects and in patients with retinal vasculopathy including hypertensive retinopathy, diabetic retinopathy, and retinal vein occlusion. Our results show that AOSLO OP motion contrast provides perfusion maps comparable to those obtained with AOSLO FA, while AOSLO OP reflectance images provide additional information such as vessel wall fine structure not as readily visible in AOSLO confocal reflectance images. AOSLO OP offers a non-invasive alternative to AOSLO FA without the need for any exogenous contrast agent.
PMCID: PMC3985984  PMID: 24761299
(110.1080) Active or adaptive optics; (290.4210) Multiple scattering; (170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology
13.  Assessing Retinal Structure In Complete Congenital Stationary Night Blindness and Oguchi Disease 
American journal of ophthalmology  2012;154(6):987-1001.e1.
To examine retinal structure and changes in photoreceptor intensity post-dark adaptation in patients with complete congenital stationary night blindness and Oguchi disease.
Prospective observational case series.
We recruited three patients with complete congenital stationary night blindness caused by mutations in GRM6, two brothers with Oguchi disease caused by mutations in GRK1, and one normal control. Retinal thickness was measured from optical coherence tomography (OCT) images. Integrity of the rod and cone mosaic was assessed using adaptive optics scanning light ophthalmoscopy. We imaged five of the patients following a period of dark adaptation, and examined layer reflectivity on OCT in a patient with Oguchi disease under light- and dark-adapted conditions.
Retinal thickness was reduced in the parafoveal region in patients with GRM6 mutations, as a result of decreased thickness of the inner retinal layers. All patients had normal photoreceptor density at all locations analyzed. Upon removal from dark adaptation, the intensity of the rods (but not cones) in the patients with Oguchi disease gradually and significantly increased. In one Oguchi patient, the outer segment layer contrast on OCT was fourfold higher under dark-adapted versus light-adapted conditions.
The selective thinning of the inner retinal layers in patients with GRM6 mutations suggests either reduced bipolar/ganglion cell numbers or altered synaptic structure in the inner retina. Our finding that rods, but not cones, change intensity after dark adaptation suggests that fundus changes in Oguchi disease are due to changes within the rods as opposed to changes at a different retinal locus.
PMCID: PMC3498541  PMID: 22959359
14.  First-order design of a reflective viewfinder for adaptive optics ophthalmoscopy 
Optics Express  2012;20(24):26596-26605.
Adaptive optics (AO) ophthalmoscopes with small fields of view have limited clinical utility. We propose to address this problem in reflective instruments by incorporating a viewfinder pupil relay designed by considering pupil and image centering and conjugation. Diverting light from an existing pupil optical relay to the viewfinder relay allows switching field of view size. Design methods that meet all four centering and conjugation conditions using either a single concave mirror or with two concave mirrors forming an off-axis afocal telescope are presented. Two different methods for calculating the focal length and orientation of the concave mirrors in the afocal viewfinder relay are introduced. Finally, a 2.2 × viewfinder mode is demonstrated in an AO scanning light ophthalmoscope.
PMCID: PMC3601596  PMID: 23187514
(080.4035) Mirror system design; (110.1080) Active or adaptive optics; (170.3890) Medical optics instrumentation; (170.4460) Ophthalmic optics and devices
15.  In vivo imaging of retinal pigment epithelium cells in age related macular degeneration 
Biomedical Optics Express  2013;4(11):2527-2539.
Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan’s method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm2, which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.
PMCID: PMC3829547  PMID: 24298413
(110.1080) Active or adaptive optics; (330.5310) Vision - photoreceptors; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
16.  In vivo dark-field imaging of the retinal pigment epithelium cell mosaic 
Biomedical Optics Express  2013;4(9):1710-1723.
Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for improving the visualization of the RPE cell boundaries including: pinhole diameter, filament thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus, wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light exposures relative to light safety standards and it is more comfortable for the subject than the traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening and monitoring disease progression.
PMCID: PMC3771842  PMID: 24049692
(170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering; (110.1080) Active or adaptive optics
17.  In vivo imaging of human retinal microvasculature using adaptive optics scanning light ophthalmoscope fluorescein angiography 
Biomedical Optics Express  2013;4(8):1305-1317.
The adaptive optics scanning light ophthalmoscope (AOSLO) allows visualization of microscopic structures of the human retina in vivo. In this work, we demonstrate its application in combination with oral and intravenous (IV) fluorescein angiography (FA) to the in vivo visualization of the human retinal microvasculature. Ten healthy subjects ages 20 to 38 years were imaged using oral (7 and/or 20 mg/kg) and/or IV (500 mg) fluorescein. In agreement with current literature, there were no adverse effects among the patients receiving oral fluorescein while one patient receiving IV fluorescein experienced some nausea and heaving. We determined that all retinal capillary beds can be imaged using clinically accepted fluorescein dosages and safe light levels according to the ANSI Z136.1-2000 maximum permissible exposure. As expected, the 20 mg/kg oral dose showed higher image intensity for a longer period of time than did the 7 mg/kg oral and the 500 mg IV doses. The increased resolution of AOSLO FA, compared to conventional FA, offers great opportunity for studying physiological and pathological vascular processes.
PMCID: PMC3756583  PMID: 24009994
(110.1080) Active or adaptive optics; (330.5380) Physiology; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
18.  The Effect of Cone Opsin Mutations on Retinal Structure and the Integrity of the Photoreceptor Mosaic 
To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations.
Eleven subjects were recruited, eight of whom have been previously described. Cone and rod density was measured using images of the photoreceptor mosaic obtained from an adaptive optics scanning light ophthalmoscope (AOSLO). Total retinal thickness, inner retinal thickness, and outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness were measured using cross-sectional spectral-domain optical coherence tomography (SD-OCT) images. Molecular genetic analyses were performed to characterize the OPN1LW/OPN1MW gene array.
While disruptions in retinal lamination and cone mosaic structure were observed in all subjects, genotype-specific differences were also observed. For example, subjects with “L/M interchange” mutations resulting from intermixing of ancestral OPN1LW and OPN1MW genes had significant residual cone structure in the parafovea (∼25% of normal), despite widespread retinal disruption that included a large foveal lesion and thinning of the parafoveal inner retina. These subjects also reported a later-onset, progressive loss of visual function. In contrast, subjects with the C203R missense mutation presented with congenital blue cone monochromacy, with retinal lamination defects being restricted to the ONL+HFL and the degree of residual cone structure (8% of normal) being consistent with that expected for the S-cone submosaic.
The photoreceptor phenotype associated with OPN1LW and OPN1MW mutations is highly variable. These findings have implications for the potential restoration of visual function in subjects with opsin mutations. Our study highlights the importance of high-resolution phenotyping to characterize cellular structure in inherited retinal disease; such information will be critical for selecting patients most likely to respond to therapeutic intervention and for establishing a baseline for evaluating treatment efficacy.
Subjects with OPN1LW and OPN1MW mutations showed a spectrum of retinal phenotypes with genotype-specific differences. This has implications for restoration of visual function in these subjects and highlights high-resolution retinal imaging as a complementary tool for emerging therapeutic efforts.
PMCID: PMC3816954  PMID: 23139274
19.  Automatic cone photoreceptor segmentation using graph theory and dynamic programming 
Biomedical Optics Express  2013;4(6):924-937.
Geometrical analysis of the photoreceptor mosaic can reveal subclinical ocular pathologies. In this paper, we describe a fully automatic algorithm to identify and segment photoreceptors in adaptive optics ophthalmoscope images of the photoreceptor mosaic. This method is an extension of our previously described closed contour segmentation framework based on graph theory and dynamic programming (GTDP). We validated the performance of the proposed algorithm by comparing it to the state-of-the-art technique on a large data set consisting of over 200,000 cones and posted the results online. We found that the GTDP method achieved a higher detection rate, decreasing the cone miss rate by over a factor of five.
PMCID: PMC3675871  PMID: 23761854
(100.0100) Image processing; (170.4470) Ophthalmology; (110.1080) Active or adaptive optics
20.  Subclinical Photoreceptor Disruption in Response to Severe Head Trauma 
Archives of Ophthalmology  2012;130(3):400-402.
PMCID: PMC3329123  PMID: 22411676
Spectral-Domain Optical Coherence Tomography; Ocular Trauma; Commotio Retinae; Adaptive Optics; Photoreceptors; Retina
21.  Retinal Crystals in Type 2 Idiopathic Macular Telangiectasia 
Ophthalmology  2011;118(12):2461-2467.
To characterize the phenotype and investigate the associations of intraretinal crystalline deposits in a large cohort of Type 2 Idiopathic Macular Telangiectasia (MacTel)
Case-control study
Patients with and without retinal crystals from the Macular Telangiectasia Project, an international multi-centre prospective study of Type 2 MacTel.
Grading of stereoscopic 30° colour fundus (CF), confocal blue light reflectance (CBR), red-free (RF) and infrared (IR) images was performed according to the MacTel Natural History Study protocol and staged using the classification system devised by Gass & Blodi. SD-OCT and adaptive optics imaging were used for a finer analysis of the phenotype. Associations between crystals and other characteristics of the disease as well as potential risk factors were investigated.
Main outcome measures
Presence of crystals, fundus signs of MacTel, clinical characteristics, presence of potential risk factors of MacTel.
Out of 443 probands enrolled in the MacTel study, 203 (46%) had crystalline deposits present; 60% of the cases were bilateral at baseline. Eyes with crystals had a mean letter score of 70.7 (SD=15.9) while those without crystals had a mean of 66.5 letters (SD=15.5, p<0.001). Crystals were present at all stages of the disease and showed high reflectivity within a wide wavelength range. They were located at the anterior surface of the nerve fibre layer, arranged along the nerve fibres, within an annular area centred on the fovea. Significant associations of crystalline deposits were found with a loss of retinal transparency, MPOD loss, fluorescein leakage, retinal thickness and a break in the IS/OS junction line. Associations with environmental risk factors were not found.
Intraretinal crystals are a frequent phenomenon associated with type 2 MacTel, they may appear at all stages and may aid in the early diagnosis of the disease. Their morphology further implicates Müller cells in the pathogenesis of the disease. Insight into their physical and chemical properties may provide clues to the metabolic pathways involved in the pathogenesis of the disease.
PMCID: PMC3433242  PMID: 21839520
22.  Relationship between the Foveal Avascular Zone and Foveal Pit Morphology 
Using a combination of in vivo retinal imaging tools, the authors found extensive variation in the size of the foveal pit and the foveal avascular zone, with larger foveal pits associated with larger foveal avascular zones.
To assess the relationship between foveal pit morphology and size of the foveal avascular zone (FAZ).
Forty-two subjects were recruited. Volumetric images of the macula were obtained using spectral domain optical coherence tomography. Images of the FAZ were obtained using either a modified fundus camera or an adaptive optics scanning light ophthalmoscope. Foveal pit metrics (depth, diameter, slope, volume, and area) were automatically extracted from retinal thickness data, whereas the FAZ was manually segmented by two observers to extract estimates of FAZ diameter and area.
Consistent with previous reports, the authors observed significant variation in foveal pit morphology. The average foveal pit volume was 0.081 mm3 (range, 0.022 to 0.190 mm3). The size of the FAZ was also highly variable between persons, with FAZ area ranging from 0.05 to 1.05 mm2 and FAZ diameter ranging from 0.20 to 1.08 mm. FAZ area was significantly correlated with foveal pit area, depth, and volume; deeper and broader foveal pits were associated with larger FAZs.
Although these results are consistent with predictions from existing models of foveal development, more work is needed to confirm the developmental link between the size of the FAZ and the degree of foveal pit excavation. In addition, more work is needed to understand the relationship between these and other anatomic features of the human foveal region, including peak cone density, rod-free zone diameter, and Henle fiber layer.
PMCID: PMC3339921  PMID: 22323466
23.  Adaptive optics scanning ophthalmoscopy with annular pupils 
Biomedical Optics Express  2012;3(7):1647-1661.
Annular apodization of the illumination and/or imaging pupils of an adaptive optics scanning light ophthalmoscope (AOSLO) for improving transverse resolution was evaluated using three different normalized inner radii (0.26, 0.39 and 0.52). In vivo imaging of the human photoreceptor mosaic at 0.5 and 10° from fixation indicates that the use of an annular illumination pupil and a circular imaging pupil provides the most benefit of all configurations when using a one Airy disk diameter pinhole, in agreement with the paraxial confocal microscopy theory. Annular illumination pupils with 0.26 and 0.39 normalized inner radii performed best in terms of the narrowing of the autocorrelation central lobe (between 7 and 12%), and the increase in manual and automated photoreceptor counts (8 to 20% more cones and 11 to 29% more rods). It was observed that the use of annular pupils with large inner radii can result in multi-modal cone photoreceptor intensity profiles. The effect of the annular masks on the average photoreceptor intensity is consistent with the Stiles-Crawford effect (SCE). This indicates that combinations of images of the same photoreceptors with different apodization configurations and/or annular masks can be used to distinguish cones from rods, even when the former have complex multi-modal intensity profiles. In addition to narrowing the point spread function transversally, the use of annular apodizing masks also elongates it axially, a fact that can be used for extending the depth of focus of techniques such as adaptive optics optical coherence tomography (AOOCT). Finally, the positive results from this work suggest that annular pupil apodization could be used in refractive or catadioptric adaptive optics ophthalmoscopes to mitigate undesired back-reflections.
PMCID: PMC3395488  PMID: 22808435
(110.1080) Active or adaptive optics; (220.1230) Apodization; (170.3880) Medical and biological imaging; (170.4460) Ophthalmic optics and devices
24.  Adaptive optics retinal imaging in the living mouse eye 
Biomedical Optics Express  2012;3(4):715-734.
Correction of the eye’s monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo.
PMCID: PMC3345801  PMID: 22574260
(170.4460) Ophthalmic optics and devices; (110.1080) Active or adaptive optics; (330.7324) Visual optics, comparative animal models
25.  Photoreceptor Structure and Function in Patients with Congenital Achromatopsia 
Assessment of retinal structure and function in achromatopsia may be useful for the selection of patients for future therapeutic trials and for monitoring therapeutic efficacy.
To assess photoreceptor structure and function in patients with congenital achromatopsia.
Twelve patients were enrolled. All patients underwent a complete ocular examination, spectral-domain optical coherence tomography (SD-OCT), full-field electroretinographic (ERG), and color vision testing. Macular microperimetry (MP; in four patients) and adaptive optics (AO) imaging (in nine patients) were also performed. Blood was drawn for screening of disease-causing genetic mutations.
Mean (±SD) age was 30.8 (±16.6) years. Mean best-corrected visual acuity was 0.85 (±0.14) logarithm of the minimal angle of resolution (logMAR) units. Seven patients (58.3%) showed either an absent foveal reflex or nonspecific retinal pigment epithelium mottling to mild hypopigmentary changes on fundus examination. Two patients showed an atrophic-appearing macular lesion. On anomaloscopy, only 5 patients matched over the entire range from 0 to 73. SD-OCT examination showed a disruption or loss of the macular inner/outer segments (IS/OS) junction of the photoreceptors in 10 patients (83.3%). Seven of these patients showed an optically empty space at the level of the photoreceptors in the fovea. AO images of the photoreceptor mosaic were highly variable but significantly disrupted from normal. On ERG testing, 10 patients (83.3%) showed evidence of residual cone responses to a single-flash stimulus response. The macular MP testing showed that the overall mean retinal sensitivity was significantly lower than normal (12.0 vs. 16.9 dB, P < 0.0001).
The current approach of using high-resolution techniques to assess photoreceptor structure and function in patients with achromatopsia should be useful in guiding selection of patients for future therapeutic trials as well as monitoring therapeutic response in these trials.
PMCID: PMC3183969  PMID: 21778272

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