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1.  In Vivo Autofluorescence Imaging of the Human and Macaque Retinal Pigment Epithelial Cell Mosaic 
Purpose
Retinal pigment epithelial (RPE) cells are critical for the health of the retina, especially the photoreceptors. A recent study demonstrated that individual RPE cells could be imaged in macaque in vivo by detecting autofluorescence with an adaptive optics scanning laser ophthalmoscope (AOSLO). The current study extended this method to image RPE cells in fixating humans in vivo and to quantify the RPE mosaic characteristics in the central retina of normal humans and macaques.
Methods
The retina was imaged simultaneously with two light channels in a fluorescence AOSLO; one channel was used for reflectance imaging of the cones while the other detected RPE autofluorescence. The excitation light was 568 nm, and emission was detected over a 40-nm range centered at 624 nm. Reflectance frames were registered to determine interframe eye motion, the motion was corrected in the simultaneously recorded autofluorescence frames, and the autofluorescence frames were averaged to give the final RPE mosaic image.
Results
In vivo imaging demonstrated that with increasing eccentricity, RPE cell density, and mosaic regularity decreased, whereas RPE cell size and spacing increased. Repeat measurements of the same retinal location 42 days apart showed the same RPE cells and distribution.
Conclusions
The RPE cell mosaic has been resolved for the first time in alert fixating human subjects in vivo using AOSLO. Mosaic analysis provides a quantitative database for studying normal and diseased RPE in vivo. This technique will allow longitudinal studies to track disease progression and assess treatment efficacy in patients and animal models of retinal disease.
doi:10.1167/iovs.08-2618
PMCID: PMC2790524  PMID: 18952914
2.  The Reduction of Retinal Autofluorescence Caused by Light Exposure 
Purpose
We have previously shown that long exposure to 568 nm light at levels below the maximum permissible exposure safety limit produces retinal damage preceded by a transient reduction in the autofluorescence of retinal pigment epithelial (RPE) cells in vivo. Here, we determine how the effects of exposure power and duration combine to produce this autofluorescence reduction and find the minimum exposure causing a detectable autofluorescence reduction.
Methods
Macaque retinas were imaged using a fluorescence adaptive optics scanning laser ophthalmoscope to resolve individual RPE cells in vivo. The retina was exposed to 568 nm light over a square subtending 0.5° with energies ranging from 1 J/cm2 to 788 J/cm2, where power and duration were independently varied.
Results
In vivo exposures of 5 J/cm2 and higher caused an immediate decrease in autofluorescence followed by either full autofluorescence recovery (exposures ≤ 210 J/cm2) or permanent RPE cell damage (exposures ≥ 247 J/cm2). No significant autofluorescence reduction was observed for exposures of 2 J/cm2 and lower. Reciprocity of exposure power and duration held for the exposures tested, implying that the total energy delivered to the retina, rather than its distribution in time, determines the amount of autofluorescence reduction.
Conclusions
That reciprocity holds is consistent with a photochemical origin, which may or may not cause retinal degeneration. The implementation of safe methods for delivering light to the retina requires a better understanding of the mechanism causing autofluorescence reduction. Finally, RPE imaging was demonstrated using light levels that do not cause a detectable reduction in autofluorescence.
doi:10.1167/iovs.09-3643
PMCID: PMC2790527  PMID: 19628734
3.  The Bisretinoids of Retinal Pigment Epithelium 
The retina exhibits an inherent autofluorescence that is imaged ophthalmoscopically as fundus autofluorescence. In clinical settings, fundus autofluorescence examination aids in the diagnosis and follow-up of many retinal disorders. Fundus autofluorescence originates from the complex mixture of bisretinoid fluorophores that are amassed by retinal pigment epithelial (RPE) cells as lipofuscin. Unlike the lipofuscin found in other cell-types, this material does not form as a result of oxidative stress. Rather, the formation is attributable to non-enzymatic reactions of vitamin A aldehyde in photoreceptor cells; transfer to RPE occurs upon phagocytosis of photoreceptor outer segments. These fluorescent pigments accumulate even in healthy photoreceptor cells and are generated as a consequence of the light capturing function of the cells. Nevertheless, the formation of this material is accelerated in some retinal disorders including recessive Stargardt disease and ELOVL-4-related retinal degeneration. As such, these bisretinoid side-products are implicated in the disease processes that threaten vision. In this article, we review our current understanding of the composition of RPE lipofuscin, the structural characteristics of the various bisretinoids, their related spectroscopic features and the biosynthetic pathways by which they form. We will revisit factors known to influence the extent of the accumulation and therapeutic strategies being used to limit bisretinoid formation. Given their origin from vitamin A aldehyde, an isomer of the visual pigment chromophore, it is not surprising that the bisretinoids of retina are light sensitive molecules. Accordingly, we will discuss recent findings that implicate the photodegradation of bisretinoid in the etiology of age-related macular degeneration.
doi:10.1016/j.preteyeres.2011.12.001
PMCID: PMC3288746  PMID: 22209824
A2E; all-trans-retinal; bisretinoid; retinal pigment epithelium; macular degeneration; retina
4.  Autofluorescence Imaging for Diagnosis and Follow-up of Cystoid Macular Edema 
Lipofuscin results from digestion of photoreceptor outer segments by the retinal pigment epithelium (RPE) and is the principal compound that causes RPE fluorescence during autofluorescence imaging. Absorption of the 488-nanometer blue light by macular pigments, especially by the carotenoids lutein and zeaxanthin, causes normal macular hypo-autofluorescence. Fundus autofluorescence imaging is being increasingly employed in ophthalmic practice to diagnose and monitor patients with a variety of retinal disorders. In macular edema for example, areas of hyper-autofluorescence are usually present which are postulated to be due to dispersion of macular pigments by pockets of intraretinal fluid. For this reason, the masking effect of macular pigments is reduced and the natural autofluorescence of lipofuscin can be observed without interference. In cystic types of macular edema, e.g. cystoid macular edema due to retinal vein occlusion, diabetic macular edema and post cataract surgery, hyper-autofluorescent regions corresponding to cystic spaces of fluid accumulation can be identified. In addition, the amount of hyper-autofluorescence seems to correspond to the severity of edema. Hence, autofluorescence imaging, as a noninvasive technique, can provide valuable information on cystoid macular edema in terms of diagnosis, follow-up and efficacy of treatment.
PMCID: PMC3520597  PMID: 23264870
Autofluorescence; Cystoid Macular Edema; Lipofuscin
5.  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.
doi:10.1364/BOE.4.001710
PMCID: PMC3771842  PMID: 24049692
(170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering; (110.1080) Active or adaptive optics
6.  Foveal Avascular Zone and Its Relationship to Foveal Pit Shape 
Optometry and Vision Science  2012;89(5):602-610.
Purpose
To investigate the retinal microvasculature at the fovea and peripheral retina in humans using the adaptive optics scanning laser ophthalmoscope (AOSLO). To examine the association of foveal avascular zone (FAZ) and foveal pit morphology.
Methods
Retinal imaging of the foveal capillary network was performed on 11 subjects (15 eyes; age range 20–54) with an AOSLO. Standard deviation maps of the AOSLO images were generated from ~10–30 frames, producing high resolution maps delineating the complete capillary distribution of the retina. Foveal pit morphology was investigated in the same subjects by using a spectral domain optical coherence tomography (SDOCT). In an additional subject, only a relatively large retinal vasculature map was obtained using AOSLO.
Results
A well demarcated FAZ was seen in 11 subjects tested with foveal capillary imaging. There was considerable individual variation in the size and shape of the FAZ. The mean FAZ area and mean FAZ effective diameter were 0.33mm2 and 622μm, respectively. Foveal thickness was found to be negatively correlated with the FAZ effective diameter.
Conclusions
The structure of the capillary network could be evaluated in the fovea and parafovea using our approach. We find that a smaller FAZ is associated with a narrower foveal pit opening and a thicker fovea.
doi:10.1097/OPX.0b013e3182504227
PMCID: PMC3348263  PMID: 22426172
foveal avascular zone; foveal pit morphology; adaptive optics scanning laser ophthalmoscope; spectral domain optical coherence tomography; retina; retinal thickness
7.  A Clinical Planning Module for Adaptive Optics SLO Imaging 
Optometry and Vision Science  2012;89(5):593-601.
Purpose
To develop a clinical planning module (CPM) to improve the efficiency of imaging subjects with a steerable wide-field adaptive optics scanning laser ophthalmoscope (AOSLO). To evaluate the performance of this module by imaging the retina in healthy and diseased eyes.
Methods
We developed a software-based CPM with two sub-modules: a navigation module and a montage acquisition module. The navigation module guides the AOSLO to image identified retinal regions from a clinical imaging platform using a matrix-based mapping between the two. The montage acquisition module systematically moves the AOSLO steering mirrors across the retina in predefined patterns. The CPM was calibrated using a model eye and tested on five normal subjects and one patient with a retinal nerve fiber layer (RNFL) defect.
Results
Within the central +/−7 degrees from the fixation target, the CPM can direct the AOSLO beam to the desired regions with localization errors of less than 0.3 degrees. The navigation error increases with eccentricity, and larger errors (up to 0.8 degrees) were evident for regions beyond 7 degrees. The repeatability of CPM navigation was tested on the same locations from 2 subjects. The localization errors between trials on different days did not differ significantly (p >0.05). The region with a size of approximately 13 by 10 degrees can be imaged in about 30minutes. An approximately 12 × 4.5 degree montage of the diseased region from a patient was imaged in 18 minutes.
Conclusions
We have implemented a clinical planning module to accurately guide the AOSLO imaging beam to desired locations, and to quickly acquire high resolution AOSLO montages. The approach is not only friendly for patients and clinicians, but also convenient to relate the imaging data between different imaging platforms.
doi:10.1097/OPX.0b013e318253e081
PMCID: PMC3348407  PMID: 22488269
adaptive optics; scanning laser ophthalmoscope; retinal nerve fiber layer; clinical planning module
8.  The susceptibility of the retina to photochemical damage from visible light 
The photoreceptor/RPE complex must maintain a delicate balance between maximizing the absorption of photons for vision and retinal image quality while simultaneously minimizing the risk of photodamage when exposed to bright light. We review the recent discovery of two new effects of light exposure on the photoreceptor/RPE complex in the context of current thinking about the causes of retinal phototoxicity. These effects are autofluorescence photobleaching in which exposure to bright light reduces lipofuscin autofluorescence and, at higher light levels, RPE disruption in which the pattern of autofluorescence is permanently altered following light exposure. Both effects occur following exposure to visible light at irradiances that were previously thought to be safe. Photopigment, retinoids involved in the visual cycle, and bisretinoids in lipofuscin have been implicated as possible photosensitizers for photochemical damage. The mechanism of RPE disruption may follow either of these paths. On the other hand, autofluorescence photobleaching is likely an indicator of photooxidation of lipofuscin. The permanent changes inherent in RPE disruption might require modification of the light safety standards. AF photobleaching recovers after several hours although the mechanisms by which this occurs are not yet clear. Understanding the mechanisms of phototoxicity is all the more important given the potential for increased susceptibility in the presence of ocular diseases that affect either the visual cycle and/or lipofuscin accumulation. In addition, knowledge of photochemical mechanisms can improve our understanding of some disease processes that may be influenced by light exposure, such as some forms of Leber’s congenital amaurosis, and aid in the development of new therapies. Such treatment prior to intentional light exposures, as in ophthalmic examinations or surgeries, could provide an effective preventative strategy.
doi:10.1016/j.preteyeres.2011.11.001
PMCID: PMC3242847  PMID: 22085795
Phototoxicity; Photochemical; Retina; Retinal pigment epithelium; Autofluorescence; Visual cycle; Lipofuscin; Bisretinoids
9.  Loss of Synchronized Retinal Phagocytosis and Age-related Blindness in Mice Lacking αvβ5 Integrin 
The Journal of Experimental Medicine  2004;200(12):1539-1545.
Daily phagocytosis by the retinal pigment epithelium (RPE) of spent photoreceptor outer segment fragments is critical for vision. In the retina, early morning circadian photoreceptor rod shedding precedes synchronized uptake of shed photoreceptor particles by RPE cells. In vitro, RPE cells use the integrin receptor αvβ5 for particle binding. Here, we tested RPE phagocytosis and retinal function in β5 integrin–deficient mice, which specifically lack αvβ5 receptors. Retinal photoresponses severely declined with age in β5−/− mice, whose RPE accumulated autofluorescent storage bodies that are hallmarks of human retinal aging and disease. β5−/− RPE in culture failed to take up isolated photoreceptor particles. β5−/− RPE in vivo retained basal uptake levels but lacked the burst of phagocytic activity that followed circadian photoreceptor shedding in wild-type RPE. Rhythmic activation of focal adhesion and Mer tyrosine kinases that mediate wild-type retinal phagocytosis was also completely absent in β5−/− retina. These results demonstrate an essential role for αvβ5 integrin receptors and their downstream signaling pathways in synchronizing retinal phagocytosis. Furthermore, they identify the β5−/− integrin mouse strain as a new animal model of age-related retinal dysfunction.
doi:10.1084/jem.20041447
PMCID: PMC2211990  PMID: 15596525
circadian rhythm; knockout; photoreceptors; retinal pigment; epithelium; vision
10.  In vivo imaging of microscopic structures in the rat retina 
Purpose
The ability to resolve single retinal cells in rodents in vivo has applications in rodent models of the visual system and retinal disease. We have characterized the performance of a fluorescence adaptive optics scanning laser ophthalmoscope (fAOSLO) that provides cellular and subcellular imaging of rat retina in vivo.
Methods
Green fluorescent protein (eGFP) was expressed in retinal ganglion cells of normal Sprague Dawley rats via intravitreal injections of adeno-associated viral vectors. Simultaneous reflectance and fluorescence retinal images were acquired using the fAOSLO. fAOSLO resolution was characterized by comparing in vivo images with subsequent imaging of retinal sections from the same eyes using confocal microscopy.
Results
Retinal capillaries and eGFP-labeled ganglion cell bodies, dendrites, and axons were clearly resolved in vivo with adaptive optics (AO). AO correction reduced the total root mean square wavefront error, on average, from 0.30 μm to 0.05 μm (1.7-mm pupil). The full width at half maximum (FWHM) of the average in vivo line-spread function (LSF) was ∼1.84 μm, approximately 82% greater than the FWHM of the diffraction-limited LSF.
Conclusions
With perfect aberration compensation, the in vivo resolution in the rat eye could be ∼2× greater than that in the human eye due to its large numerical aperture (∼0.43). While the fAOSLO corrects a substantial fraction of the rat eye's aberrations, direct measurements of retinal image quality reveal some blur beyond that expected from diffraction. Nonetheless, subcellular features can be resolved, offering promise for using AO to investigate the rodent eye in vivo with high resolution.
doi:10.1167/iovs.09-3675
PMCID: PMC2873188  PMID: 19578019
11.  Imaging of Vascular Wall Fine Structure in the Human Retina Using Adaptive Optics Scanning Laser Ophthalmoscopy 
Purpose.
To improve the ability to image the vascular walls in the living human retina using multiply-scattered light imaging with an adaptive optics scanning laser ophthalmoscope (AOSLO).
Methods.
In vivo arteriolar wall imaging was performed on eight healthy subjects using the Indiana AOSLO. Noninvasive imaging of vascular mural cells and wall structure were performed using systematic control of the position of a 10× Airy disk confocal aperture. Retinal arteries and arterioles were divided into four groups based on their lumen diameters (group 1: ≥100 μm; group 2: 50–99 μm; group 3: 10–49 μm; group 4: <10 μm).
Results.
Fine structure of retinal vasculature and scattering behavior of erythrocytes were clearly visualized in all eight subjects. In group 1 vessels the mural cells were flatter and formed the outer layer of regularly spaced cells of a two (or more) layered vascular wall. In the vessels of groups 2 and 3, mural cells were visualized as distinct cells lying along the lumen of the blood vessel, resulting in a wall of irregular thickness. Vascular wall components were not readily identified in group 4 vessels.
Conclusions.
Our results show that retinal vascular mural cells and wall structure can be readily resolved in healthy subjects using AOSLO with multiply scattered light imaging for retinal vessels with a lumen diameter greater than or equal to 10 μm. Our noninvasive imaging approach allows direct assessment of the cellular structure of the vascular wall in vivo with potential applications in retinal vascular diseases such as diabetes and hypertension.
We investigated the ability of retinal imaging with an adaptive optics scanning laser ophthalmoscope combined with a dark field detection scheme to improve the visualization of the vascular walls with the goal of enabling cellular imaging of the retinal vasculature.
doi:10.1167/iovs.13-13027
PMCID: PMC3813321  PMID: 24071955
retina; blood vessels; pericytes; endothelial cells; adaptive optics scanning laser ophthalmoscope; dark field imaging
12.  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.
doi:10.1364/BOE.4.001305
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
13.  Cone Photoreceptor Abnormalities Correlate with Vision Loss in Patients with Stargardt Disease 
Adaptive optics scanning laser ophthalmoscopy demonstrated abnormal cone spacing in regions of abnormal fundus autofluorescence and reduced visual function in 12 patients with Stargardt disease.
Purpose.
To study the relationship between macular cone structure, fundus autofluorescence (AF), and visual function in patients with Stargardt disease (STGD).
Methods.
High-resolution images of the macula were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography in 12 patients with STGD and 27 age-matched healthy subjects. Measures of retinal structure and AF were correlated with visual function, including best-corrected visual acuity, color vision, kinetic and static perimetry, fundus-guided microperimetry, and full-field electroretinography. Mutation analysis of the ABCA4 gene was completed in all patients.
Results.
Patients were 15 to 55 years old, and visual acuity ranged from 20/25–20/320. Central scotomas were present in all patients, although the fovea was spared in three patients. The earliest cone spacing abnormalities were observed in regions of homogeneous AF, normal visual function, and normal outer retinal structure. Outer retinal structure and AF were most normal near the optic disc. Longitudinal studies showed progressive increases in AF followed by reduced AF associated with losses of visual sensitivity, outer retinal layers, and cones. At least one disease-causing mutation in the ABCA4 gene was identified in 11 of 12 patients studied; 1 of 12 patients showed no disease-causing ABCA4 mutations.
Conclusions.
AOSLO imaging demonstrated abnormal cone spacing in regions of abnormal fundus AF and reduced visual function. These findings provide support for a model of disease progression in which lipofuscin accumulation results in homogeneously increased AF with cone spacing abnormalities, followed by heterogeneously increased AF with cone loss, then reduced AF with cone and RPE cell death. (ClinicalTrials.gov number, NCT00254605.)
doi:10.1167/iovs.10-6538
PMCID: PMC3109028  PMID: 21296825
14.  Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability 
Cell Death & Disease  2013;4(8):e781-.
Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood–retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side – that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.
doi:10.1038/cddis.2013.303
PMCID: PMC3763463  PMID: 23990021
neurodegeneration; photoreceptor; VEGF; retina; gene therapy
15.  AUTOFLUORESCENCE IMAGING FINDINGS IN LONG-STANDING CHORIORETINAL FOLDS 
Retinal cases & brief reports  2009;3(2):137-139.
Background
Chorioretinal folds typically involve the choroid, Bruch membrane, retinal pigment epithelium (RPE), and sometimes overlying neurosensory retina. von Winning hypothesized that the alternate banding pattern of choroidal folds shown by fluorescein angiography is explained by RPE density. To our knowledge, autofluorescence imaging of chorioretinal folds has not been previously described.
Methods
Case report.
Patient
A 47-year-old healthy hyperopic man had best-corrected visual acuity of 20/30 in the right eye and 20/25 in the left eye. Posterior segment examination revealed bilateral chorioretinal folds with subtle streaks of RPE hyperpigmentation and hypopigmentation emanating from both optic nerve heads.
Results
Early-phase fluorescein angiography revealed the characteristic pattern of alternating light and dark bands. Autofluorescence imaging disclosed a similar pattern as well as peripapillary mottling. The alternating patterns of light and dark bands observed using autofluorescence imaging and fluorescein angiography were found to be precisely in register but inverted.
Conclusions
Autofluorescence imaging noninvasively demonstrates the pathognomonic pattern of alternating light and dark bands shown by fluorescein angiography diagnostic of choroidal folds but in an inverse fashion. This observation provides independent support of von Winning’s hypothesis regarding the etiopathogenesis of the banding pattern.
doi:10.1097/ICB.0b013e3181679f91
PMCID: PMC2830013  PMID: 20198126
chorioretinal folds; autofluorescence; fluorescein angiography; hyperopia; retinal pigment epithelium
16.  Fundus Autofluorescence in the Abca4−/− Mouse Model of Stargardt Disease—Correlation With Accumulation of A2E, Retinal Function, and Histology 
Purpose.
To investigate fundus autofluorescence (AF) characteristics in the Abca4−/− mouse, an animal model for AMD and Stargardt disease, and to correlate findings with functional, structural, and biochemical assessments.
Methods.
Blue (488 nm) and near-infrared (790 nm) fundus AF images were quantitatively and qualitatively analyzed in pigmented Abca4−/− mice and wild type (WT) controls in vivo. Functional, structural, and biochemical assessments included electroretinography (ERG), light and electron microscopic analysis, and A2E quantification. All assessments were performed across age groups.
Results.
In Abca4−/− mice, lipofuscin-related 488 nm AF increased early in life with a ceiling effect after 6 months. This increase was first paralleled by an accumulation of typical lipofuscin granules in the retinal pigment epithelium (RPE). Later, lipofuscin and melanin granules decreased in number, whereas melanolipofuscin granules increased. This increase in melanolipofuscin granules paralleled an increase in melanin-related 790 nm AF. Old Abca4−/− mice revealed a flecked fundus AF pattern at both excitation wavelengths. The amount of A2E, a major lipofuscin component, increased 10- to 12-fold in 6- to 9-month-old Abca4−/− mice compared with controls, while 488 nm AF intensity only increased 2-fold. Despite pronounced lipofuscin accumulation in the RPE of Abca4−/− mice, ERG and histology showed a slow age-related thinning of the photoreceptor layer similar to WT controls up to 12 months.
Conclusions.
Fundus AF can be used to monitor lipofuscin accumulation and melanin-related changes in vivo in mouse models of retinal disease. High RPE lipofuscin may not adversely affect retinal structure or function over prolonged time intervals, and melanin-related changes (melanolipofuscin formation) may occur before the decline in retinal function.
Increased fundus autofluorescence in the Abca4−/− mouse correlates to bisretinoid accumulation and alterations of melanosomes in the retinal pigment epithelium, without adverse functional effects over prolonged time intervals. The mouse phenotype parallels findings in human retinal disease.
doi:10.1167/iovs.13-11688
PMCID: PMC3747716  PMID: 23761084
fundus autofluorescence; mouse model; Abca4; A2E
17.  In vivo adaptive optics microvascular imaging in diabetic patients without clinically severe diabetic retinopathy 
Biomedical Optics Express  2014;5(3):961-974.
We used a confocal adaptive optics scanning laser ophthalmoscope (AOSLO) to image the retina of subjects with non-proliferative diabetic retinopathy (NPDR). To improve visualization of different retinal features, the size and alignment of the confocal aperture were varied. The inner retinal layers contained clearly visualized retinal vessels. In diabetic subjects there was extensive capillary remodeling despite the subjects having only mild or moderate NPDR. Details of the retinal microvasculature were readily imaged with a larger confocal aperture. Hard exudates were observed with the AOSLO in all imaging modes. Photoreceptor layer images showed regions of bright cones and dark areas, corresponding in location to overlying vascular abnormalities and retinal edema. Clinically undetected intraretinal vessel remodeling and varying blood flow patterns were found. Perifoveal capillary diameters were larger in the diabetic subjects (p<0.01), and small arteriolar walls were thickened, based on wall to lumen measurements (p<.05). The results suggest that existing clinical classifications based on lower magnification clinical assessment may not adequately measure key vascular differences among individuals with NPDR.
doi:10.1364/BOE.5.000961
PMCID: PMC3959854
(170.3880) Medical and biological imaging; (170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology
18.  Epiretinal membrane surgery for combined hamartoma of the retina and retinal pigment epithelium: role of multimodal analysis 
Background
The purpose of this study was to evaluate the role of spectral domain optical coherence tomography (SD-OCT), MP-1 microperimetry, and fundus autofluorescence imaging for planning surgical procedures in combined hamartomas of the retina and retinal pigment epithelium (CHR-RPE) and following epiretinal membrane removal.
Methods
In an interventional retrospective case series, six consecutive subjects with CHR-RPE underwent vitrectomy and epiretinal membrane peeling, with 4 years of follow-up. Each underwent complete ophthalmic examination, including best corrected visual acuity, fundus examination, fundus fluorescein angiography, SD-OCT, MP-1, and fundus autofluorescence at one, 6, 12, and 48 months.
Results
Six eyes from six subjects with CHR-RPE were studied (mean age 31 ± 14 years). All patients were phakic and five were male (83.3%). Lesions were unilateral, ie, three macular, two juxtapapillary and macular, and one pericentral. Preoperative best corrected visual acuity was 0.3 ± 0.08 Snellen, with significant improvement to 0.9 ± 0.17 Snellen (P = 0.001) at 4 years of follow-up. Mean retinal sensitivity within the central 20° field improved from 16.6 ± 1.84 dB to 18.8 ± 0.96 dB (P = 0.07). There was also a statistically significant reduction in the visual defect (P = 0.04). SD-OCT demonstrated that the epiretinal membranes were completely removed in all but one patient, with significantly decreased macular edema on follow-up at one, 6, 12, and 48 months (P = 0.001). A positive correlation was shown between preoperative macular sensitivity and postoperative best corrected visual acuity. Fundus autofluorescence demonstrated a block in background autofluorescence at the site of the lesion, and hyperautofluorescence at the edematous retina overlain by the epiretinal membrane.
Conclusion
Surgery is an effective treatment for CHR-RPE. SD-OCT, fundus autofluorescence, and MP-1 are valuable and noninvasive tools to guide surgical procedures for CHR-RPE. To the best of our knowledge, this study represents the first use of MP-1 in CHR-RPE in conjunction with SD-OCT and fundus autofluorescence imaging for better guided surgery as well as anatomical and functional prognosis.
doi:10.2147/OPTH.S39909
PMCID: PMC3553654  PMID: 23378735
vitrectomy; epiretinal membrane; combined hamartoma of the retina and retinal pigment epithelium
19.  Subclinical Capillary Changes in Non Proliferative Diabetic Retinopathy 
Optometry and Vision Science  2012;89(5):E692-E703.
Purpose
To establish adaptive optics scanning laser ophthalmoscopy (AOSLO) as a method to detect and characterize microscopic signs of diabetic retinopathy, in capillaries and cone photoreceptors in the parafovea.
Methods
Recently, AOSLO has enabled noninvasive assessment of photoreceptors, capillaries, and leukocytes in the retinas of live human subjects. Repeated application of AOSLO imaging along with comparison to fluorescein angiography was used to track individual capillaries near the foveal avascular zone (FAZ) from one eye affected with severe non-proliferative diabetic retinopathy. Fluorescein angiography was used to identify clinical signs of diabetic retinopathy, such as microaneurysms and intra-retinal microvascular abnormalities (IRMAs), and corresponding regions were imaged and assessed using the AOSLO. In addition, the structural integrity of photoreceptors and the spatial distribution of leukocytes around the parafoveal capillary network were quantitatively assessed.
Results
Capillaries and cone photoreceptors were visualized using the AOSLO, without the use of injected contrast agents. Although the majority of capillaries were stable over a period of 16 months, one capillary at the edge of the FAZ dropped out, leading to a small, but significant increase in FAZ size. Longitudinal assessment of the capillaries also showed microaneurysm formation and disappearance, as well as the formation of tiny capillary bends similar in appearance to IRMAs. The leukocytes in the capillary network were found to preferentially travel through the same routes in all four visits, suggesting that these channels are robust against small changes to the surrounding capillaries. In this eye, cone photoreceptor spacing was increased in the fovea when compared to normal data, but stable across all visits.
Conclusions
AOSLO imaging can be used to longitudinally track capillaries, leukocytes and photoreceptors in diabetic retinopathy. Capillary changes that can be detected include dropout of individual capillaries, as well as formation and disappearance of microaneurysms.
doi:10.1097/OPX.0b013e3182548b07
PMCID: PMC3348374  PMID: 22525131
adaptive optics; capillaries; diabetic retinopathy; leukocyte; microaneurysm; photoreceptors
20.  Adaptive optics scanning laser ophthalmoscope with integrated wide-field retinal imaging and tracking 
We have developed a new, unified implementation of the adaptive optics scanning laser ophthalmoscope (AOSLO) incorporating a wide-field line-scanning ophthalmoscope (LSO) and a closed-loop optical retinal tracker. AOSLO raster scans are deflected by the integrated tracking mirrors so that direct AOSLO stabilization is automatic during tracking. The wide-field imager and large-spherical-mirror optical interface design, as well as a large-stroke deformable mirror (DM), enable the AOSLO image field to be corrected at any retinal coordinates of interest in a field of >25 deg. AO performance was assessed by imaging individuals with a range of refractive errors. In most subjects, image contrast was measurable at spatial frequencies close to the diffraction limit. Closed-loop optical (hardware) tracking performance was assessed by comparing sequential image series with and without stabilization. Though usually better than 10 μm rms, or 0.03 deg, tracking does not yet stabilize to single cone precision but significantly improves average image quality and increases the number of frames that can be successfully aligned by software-based post-processing methods. The new optical interface allows the high-resolution imaging field to be placed anywhere within the wide field without requiring the subject to re-fixate, enabling easier retinal navigation and faster, more efficient AOSLO montage capture and stitching.
PMCID: PMC3071649  PMID: 21045887
21.  Near-infrared light photoacoustic ophthalmoscopy 
Biomedical Optics Express  2012;3(4):792-799.
We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.
doi:10.1364/BOE.3.000792
PMCID: PMC3345807  PMID: 22574266
(110.5120) Photoacoustic imaging; (170.4470) Ophthalmology
22.  Pathological Consequences of Long-Term Mitochondrial Oxidative Stress in the Mouse Retinal Pigment Epithelium 
Experimental eye research  2012;101:60-71.
Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.
doi:10.1016/j.exer.2012.05.013
PMCID: PMC3419481  PMID: 22687918
retinal pigment epithelium; oxidative stress; superoxide dismutase; mitochondria; mouse model; age related macular degeneration
23.  Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line 
BMC Cell Biology  2009;10:33.
Background
The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.
Results
The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency.
Conclusion
The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal maculopathies.
doi:10.1186/1471-2121-10-33
PMCID: PMC3152772  PMID: 19413901
24.  In vivo Optical Coherence Tomography of Light-Driven Melanosome Translocation in Retinal Pigment Epithelium 
Scientific Reports  2013;3:2644.
Optical coherence tomography (OCT) may revolutionize fundamental investigation and clinical management of age-related macular degeneration and other eye diseases. However, quantitative OCT interpretation is hampered due to uncertain sub-cellular correlates of reflectivity in the retinal pigment epithelium (RPE) and photoreceptor. The purpose of this study was twofold: 1) to test OCT correlates in the RPE, and 2) to demonstrate the feasibility of longitudinal OCT monitoring of sub-cellular RPE dynamics. A high resolution OCT was constructed to achieve dynamic imaging of frog eyes, in which light-driven translocation of RPE melanosomes occurred within the RPE cell body and apical processes. Comparative histological examination of dark- and light-adapted eyes indicated that the RPE melanin granule, i.e., melanosome, was a primary OCT correlate. In vivo OCT imaging of RPE melanosomes opens the opportunity for quantitative assessment of RPE abnormalities associated with disease, and enables longitudinal investigation of RPE kinetics correlated with visual function.
doi:10.1038/srep02644
PMCID: PMC3770963  PMID: 24025778
25.  SLO-infrared imaging of the macula and its correlation with functional loss and structural changes in patients with Stargardt disease 
Retina (Philadelphia, Pa.)  2011;31(5):949-958.
Purpose
To correlate the degree of functional loss with structural changes in patients with Stargardt disease.
Methods
Eighteen eyes of 10 Stargardt patients were studied. Scanning laser ophthalmoscope (SLO) infrared images were compared to corresponding spectral domain optical coherence tomography (SD-OCT) scans. Additionally, SLO microperimetry was performed and results were superimposed on SLO infrared images and in selected cases on fundus autofluorescence (FAF) images.
Results
Seventeen of 18 eyes showed a distinct hypo-reflective foveal and/or perifoveal area with distinct borders on SLO-infrared images which was less evident on funduscopy and incompletely depicted in FAF images. This hypo-reflective zone corresponded to areas of significantly elevated psychophysical thresholds on microperimetry testing, in addition to thinning of the retinal pigment epithelium (RPE), disorganization or loss of the photoreceptor cell inner-outer segment (IS-OS) junction and external limiting membrane (ELM) on SD-OCT.
Conclusion
SLO-infrared fundus images are useful for depicting retinal structural changes in Stargardt patients. An SD-OCT/SLO microperimetry device allows for a direct correlation of structural abnormalities with functional defects that will likely be applicable for the determination of retinal areas for potential improvement of retinal function in these patients during future clinical trials and for the monitoring of the diseases' natural history.
doi:10.1097/IAE.0b013e3181f441f6
PMCID: PMC3116073  PMID: 21293320
microperimetry; SLO infrared imaging; Stargardt disease; fundus autofluorescence imaging

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