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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.  In vivo two-photon imaging of the mouse retina 
Biomedical Optics Express  2013;4(8):1285-1293.
Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.
doi:10.1364/BOE.4.001285
PMCID: PMC3756587  PMID: 24009992
(330.4460) Ophthalmic optics and devices; (180.4315) Nonlinear microscopy; (170.0110) Imaging systems
3.  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.
doi:10.1364/BOE.3.001647
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
4.  Spatiotemporal regulation of ATP and Ca2+ dynamics in vertebrate rod and cone ribbon synapses 
Molecular Vision  2007;13:887-919.
Purpose
In conventional neurons, Ca2+ enters presynaptic terminals during an action potential and its increased local concentration triggers transient exocytosis. In contrast, vertebrate photoreceptors are nonspiking neurons that maintain sustained depolarization and neurotransmitter release from ribbon synapses in darkness and produce light-dependent graded hyperpolarizing responses. Rods transmit single photon responses with high fidelity, whereas cones are less sensitive and exhibit faster response kinetics. These differences are likely due to variations in presynaptic Ca2+ dynamics. Metabolic coupling and cross-talk between mitochondria, endoplasmic reticulum (ER), plasma membrane Ca2+ ATPase (PMCA), and Na+-Ca2+ exchanger (NCX) coordinately control presynaptic ATP production and Ca2+ dynamics. The goal of our structural and functional studies was to determine the spatiotemporal regulation of ATP and Ca2+ dynamics in rod spherules and cone pedicles.
Methods
Central retina tissue from C57BL/6 mice was used. Laser scanning confocal microscopy (LSCM) experiments were conducted on fixed-frozen vertical sections. Primary antibodies were selected for their tissue/cellular specificity and ability to recognize single, multiple or all splice variants of selected isoforms. Electron microscopy (EM) and 3-D electron tomography (ET) studies used our standard procedures on thin- and thick-sectioned retinas, respectively. Calibrated fluo-3-Ca2+ imaging experiments of dark- and light-adapted rod and cone terminals in retinal slices were conducted.
Results
Confocal microscopy showed that mitochondria, ER, PMCA, and NCX1 exhibited distinct retinal lamination patterns and differential distribution in photoreceptor synapses. Antibodies for three distinct mitochondrial compartments differentially labeled retinal areas with high metabolic demand: rod and cone inner segments, previously undescribed cone juxtanuclear mitochondria and the two plexiform layers. Rod spherule membranes uniformly and intensely stained for PMCA, whereas the larger cone pedicles preferentially stained for NCX1 at their active zones and PMCA near their mitochondria. EM and ET revealed that mitochondria in rod spherules and cone pedicles differed markedly in their number, location, size, volume, and total cristae surface area, and cristae junction diameter. Rod spherules had one large ovoid mitochondrion located near its active zone, whereas cone pedicles averaged five medium-sized mitochondria clustered far from their active zones. Most spherules had one ribbon synapse, whereas pedicles contained numerous ribbon synapses. Fluo-3 imaging studies revealed that during darkness rod spherules maintained a lower [Ca2+] than cone pedicles, whereas during light adaptation pedicles rapidly lowered their [Ca2+] below that observed in spherules.
Conclusions
These findings indicate that ATP demand and mitochondrial ATP production are greater in cone pedicles than rod spherules. Rod spherules employ high affinity/low turnover PMCA and their mitochondrion to maintain a relatively low [Ca2+] in darkness, which increases their sensitivity and signal-to-noise ratio. In contrast, cone pedicles utilize low affinity/high turnover NCX to rapidly lower their high [Ca2+] during light adaptation, which increases their response kinetics. Spatiotemporal fluo-3-Ca2+ imaging results support our immunocytochemical results. The clustering of cone pedicle mitochondria likely provides increased protection from Ca2+ overload and permeability transition. In summary, these novel studies reveal that several integrated cellular and subcellular components interact to regulate ATP and Ca2+ dynamics in rod and cone synaptic terminals. These results should provide a greater understanding of in vivo photoreceptor synaptic terminal exocytosis/endocytosis, Ca2+ overload and therapies for retinal degenerations.
PMCID: PMC2774461  PMID: 17653034
5.  Two-photon excited autofluorescence imaging of freshly isolated frog retinas 
Biomedical Optics Express  2011;2(6):1494-1503.
The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.
doi:10.1364/BOE.2.001494
PMCID: PMC3114218  PMID: 21698013
(170.2655) Functional monitoring and imaging; (170.4580) Optical diagnostics for medicine; (170.3880) Medical and biological imaging; (330.5380) Physiology
6.  The Effect of Cone Opsin Mutations on Retinal Structure and the Integrity of the Photoreceptor Mosaic 
Purpose.
To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations.
Methods.
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.
Results.
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.
Conclusions.
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.
doi:10.1167/iovs.12-11087
PMCID: PMC3816954  PMID: 23139274
7.  Imaging retinal mosaics in the living eye 
Eye  2011;25(3):301-308.
Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.
doi:10.1038/eye.2010.221
PMCID: PMC3178316  PMID: 21390064
retina; adaptive optics; imaging, photoreceptors; retinal pigment epithelium; retinal ganglion cells
8.  Assessing Retinal Structure In Complete Congenital Stationary Night Blindness and Oguchi Disease 
American journal of ophthalmology  2012;154(6):987-1001.e1.
PURPOSE
To examine retinal structure and changes in photoreceptor intensity post-dark adaptation in patients with complete congenital stationary night blindness and Oguchi disease.
DESIGN
Prospective observational case series.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.1016/j.ajo.2012.06.003
PMCID: PMC3498541  PMID: 22959359
9.  Variation of Cone Photoreceptor Packing Density with Retinal Eccentricity and Age 
This is the first in vivo study of cone photoreceptor packing density at different ages.
Purpose.
To study the variation of cone photoreceptor packing density across the retina in healthy subjects of different ages.
Methods.
High-resolution adaptive optics scanning laser ophthalmoscope (AOSLO) systems were used to systematically image the retinas of two groups of subjects of different ages. Ten younger subjects (age range, 22–35 years) and 10 older subjects (age range, 50–65 years) were tested. Strips of cone photoreceptors, approximately 12° × 1.8° long were imaged for each of the four primary retinal meridians: superior, inferior, nasal, and temporal. Cone photoreceptors within the strips were counted, and cone photoreceptor packing density was calculated. Statistical analysis (three-way ANOVA) was used to calculate the interaction for cone photoreceptor packing density between age, meridian, and eccentricity.
Results.
As expected, cone photoreceptor packing density was higher close to the fovea and decreased with increasing retinal eccentricity from 0.18 to 3.5 mm (∼0.6–12°). Older subjects had approximately 75% of the cone density at 0.18 mm (∼0.6°), and this difference decreased rapidly with eccentricity, with the two groups having similar cone photoreceptor packing densities beyond 0.5 mm retinal eccentricity on average.
Conclusions.
Cone packing density in the living human retina decreases as a function of age within the foveal center with the largest difference being found at our most central measurement site. At all ages, the retina showed meridional difference in cone densities, with cone photoreceptor packing density decreasing faster with increasing eccentricity in the vertical dimensions than in the horizontal dimensions.
doi:10.1167/iovs.11-7199
PMCID: PMC3183974  PMID: 21724911
10.  Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina 
Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (>850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.
doi:10.3791/3457
PMCID: PMC3308615  PMID: 22105413
11.  Retinal cone mosaic imaged with transverse scanning optical coherence tomography 
Optics letters  2006;31(12):1821-1823.
We improved our recently reported retinal OCT system based on transverse priority scanning to achieve high resolution in both the transverse and the axial directions. The implementation of an additional SLO channel enables precise on-line focusing. The system enables imaging of the human retinal cone mosaic off the foveal center without adaptive optics. We demonstrate, for what is believed to be the first time, cone mosaic imaging simultaneously in the scanning laser ophthalmoscope and optical coherence tomography (OCT) channels. OCT B-scan images demonstrate that the cone mosaic is observable in two adjacent layers. Furthermore, we present what are believed to be the first C-scan OCT images of the cone mosaic and show that the major part of light backscattered from below the photoreceptor layer is not guided back toward the pupil by the photoreceptors.
PMCID: PMC2956974  PMID: 16729082
12.  In-vivo imaging of retinal nerve fiber layer vasculature: imaging - histology comparison 
BMC Ophthalmology  2009;9:9.
Background
Although it has been suggested that alterations of nerve fiber layer vasculature may be involved in the etiology of eye diseases, including glaucoma, it has not been possible to examine this vasculature in-vivo. This report describes a novel imaging method, fluorescence adaptive optics (FAO) scanning laser ophthalmoscopy (SLO), that makes possible for the first time in-vivo imaging of this vasculature in the living macaque, comparing in-vivo and ex-vivo imaging of this vascular bed.
Methods
We injected sodium fluorescein intravenously in two macaque monkeys while imaging the retina with an FAO-SLO. An argon laser provided the 488 nm excitation source for fluorescence imaging. Reflectance images, obtained simultaneously with near infrared light, permitted precise surface registration of individual frames of the fluorescence imaging. In-vivo imaging was then compared to ex-vivo confocal microscopy of the same tissue.
Results
Superficial focus (innermost retina) at all depths within the NFL revealed a vasculature with extremely long capillaries, thin walls, little variation in caliber and parallel-linked structure oriented parallel to the NFL axons, typical of the radial peripapillary capillaries (RPCs). However, at a deeper focus beneath the NFL, (toward outer retina) the polygonal pattern typical of the ganglion cell layer (inner) and outer retinal vasculature was seen. These distinguishing patterns were also seen on histological examination of the same retinas. Furthermore, the thickness of the RPC beds and the caliber of individual RPCs determined by imaging closely matched that measured in histological sections.
Conclusion
This robust method demonstrates in-vivo, high-resolution, confocal imaging of the vasculature through the full thickness of the NFL in the living macaque, in precise agreement with histology. FAO provides a new tool to examine possible primary or secondary role of the nerve fiber layer vasculature in retinal vascular disorders and other eye diseases, such as glaucoma.
doi:10.1186/1471-2415-9-9
PMCID: PMC2744910  PMID: 19698151
13.  Adaptive optics fundus images of cone photoreceptors in the macula of patients with retinitis pigmentosa 
Background
The purpose of this study was to examine cone photoreceptors in the macula of patients with retinitis pigmentosa using an adaptive optics fundus camera and to investigate any correlations between cone photoreceptor density and findings on optical coherence tomography and fundus autofluorescence.
Methods
We examined two patients with typical retinitis pigmentosa who underwent ophthalmological examination, including measurement of visual acuity, and gathering of electroretinographic, optical coherence tomographic, fundus autofluorescent, and adaptive optics fundus images. The cone photoreceptors in the adaptive optics images of the two patients with retinitis pigmentosa and five healthy subjects were analyzed.
Results
An abnormal parafoveal ring of high-density fundus autofluorescence was observed in the macula in both patients. The border of the ring corresponded to the border of the external limiting membrane and the inner segment and outer segment line in the optical coherence tomographic images. Cone photoreceptors at the abnormal parafoveal ring were blurred and decreased in the adaptive optics images. The blurred area corresponded to the abnormal parafoveal ring in the fundus autofluorescence images. Cone densities were low at the blurred areas and at the nasal and temporal retina along a line from the fovea compared with those of healthy controls. The results for cone spacing and Voronoi domains in the macula corresponded with those for the cone densities.
Conclusion
Cone densities were heavily decreased in the macula, especially at the parafoveal ring on high-density fundus autofluorescence in both patients with retinitis pigmentosa. Adaptive optics images enabled us to observe in vivo changes in the cone photoreceptors of patients with retinitis pigmentosa, which corresponded to changes in the optical coherence tomographic and fundus autofluorescence images.
doi:10.2147/OPTH.S39879
PMCID: PMC3558310  PMID: 23378739
adaptive optics fundus camera; cone photoreceptor; fundus autofluorescence; optical coherence tomography; retinitis pigmentosa
14.  Controlling gain one photon at a time 
eLife  2013;2:e00467.
Adaptation is a salient property of sensory processing. All adaptational or gain control mechanisms face the challenge of obtaining a reliable estimate of the property of the input to be adapted to and obtaining this estimate sufficiently rapidly to be useful. Here, we explore how the primate retina balances the need to change gain rapidly and reliably when photons arrive rarely at individual rod photoreceptors. We find that the weakest backgrounds that decrease the gain of the retinal output signals are similar to those that increase human behavioral threshold, and identify a novel site of gain control in the retinal circuitry. Thus, surprisingly, the gain of retinal signals begins to decrease essentially as soon as background lights are detectable; under these conditions, gain control does not rely on a highly averaged estimate of the photon count, but instead signals from individual photon absorptions trigger changes in gain.
DOI: http://dx.doi.org/10.7554/eLife.00467.001
eLife digest
To process the sights and sounds around us, our senses must be attuned to a huge range of signals: from barely audible whispers to deafening rock concerts, and from dim glimmers of light to bright spotlights. Sensory neurons face the challenge of encoding this huge range of inputs within their much more restricted response range. Thus, neurons in our eyes and ears must continually adjust their gain or sensitivity to match changes in the light and sound inputs. These gain control processes must operate rapidly to keep up with the ever-changing input signals, but must also operate accurately so as not to distort the inputs.
The trade-off between rapid and accurate gain control can be illustrated by considering how the retina processes information at low light levels. There are two main types of light-sensitive cells in the retina: rods and cones. Vision at night relies on the ability of the rods to detect single photons—the smallest unit of light. In starlight, an individual rod will register photons only rarely, and most of the time, the majority of the rods will not register any photons. Neurons in the retinal circuits that read out the rod signals receive input from hundreds or thousands of rods, and those rod inputs are highly amplified to allow detection of the responses produced when a tiny fraction of the rods absorbs a photon. But this amplification is dangerous, as it could easily saturate retinal signals when light levels increase. Gain control mechanisms are needed to avoid such saturation.
Schwartz and Rieke now add to our understanding of this process by examining how the retinas of non-human primates behave in low light. They reveal that levels of background light that can only just be detected behaviorally trigger retinal gain controls; these gain controls operate when less than 1% of rods absorb a photon. Under these conditions, the physics of light itself will cause considerable variability in the stream of photons arriving at the retina, leading to high variability in the gain of retinal responses. Nonetheless, changes in gain occurred rapidly following changes in background, indicating that the underlying mechanisms spend little time averaging incident photons. Taken together, these findings will require revisiting our ideas about how adaptational mechanisms balance the competing demands of speed and reliability to help us see the world around us.
DOI: http://dx.doi.org/10.7554/eLife.00467.002
doi:10.7554/eLife.00467
PMCID: PMC3654457  PMID: 23682314
macaque; adaptation; retinal signal processing; neural computation; Other
15.  Reflective afocal broadband adaptive optics scanning ophthalmoscope 
Biomedical Optics Express  2011;2(6):1757-1768.
A broadband adaptive optics scanning ophthalmoscope (BAOSO) consisting of four afocal telescopes, formed by pairs of off-axis spherical mirrors in a non-planar arrangement, is presented. The non-planar folding of the telescopes is used to simultaneously reduce pupil and image plane astigmatism. The former improves the adaptive optics performance by reducing the root-mean-square (RMS) of the wavefront and the beam wandering due to optical scanning. The latter provides diffraction limited performance over a 3 diopter (D) vergence range. This vergence range allows for the use of any broadband light source(s) in the 450-850 nm wavelength range to simultaneously image any combination of retinal layers. Imaging modalities that could benefit from such a large vergence range are optical coherence tomography (OCT), multi- and hyper-spectral imaging, single- and multi-photon fluorescence. The benefits of the non-planar telescopes in the BAOSO are illustrated by resolving the human foveal photoreceptor mosaic in reflectance using two different superluminescent diodes with 680 and 796 nm peak wavelengths, reaching the eye with a vergence of 0.76 D relative to each other.
doi:10.1364/BOE.2.001757
PMCID: PMC3114240  PMID: 21698035
(110.1080) Active or adaptive optics; (080.4035) Mirror system design; (170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology
16.  Macular Cone Abnormalities in Retinitis Pigmentosa with Preserved Central Vision Using Adaptive Optics Scanning Laser Ophthalmoscopy 
PLoS ONE  2013;8(11):e79447.
Purpose
To assess macular photoreceptor abnormalities in eyes with retinitis pigmentosa (RP) with preserved central vision using adaptive optics scanning laser ophthalmoscopy (AO-SLO).
Methods
Fourteen eyes of 14 patients with RP (best-corrected visual acuity 20/20 or better) and 12 eyes of 12 volunteers underwent a full ophthalmologic examination, fundus autofluorescence, spectral-domain optical coherence tomography (SD-OCT), and imaging with a prototype AO-SLO system. Cone density and spatial organization of the cone mosaic were assessed using AO-SLO images.
Results
In 3 eyes with RP and preserved central vision, cones formed a mostly regular mosaic pattern with small patchy dark areas, and in 10 eyes, the cone mosaic patterns were less regular, and large dark regions with missing cones were apparent. Only one eye with RP demonstrated a normal, regular cone mosaic pattern. In eyes with RP, cone density was significantly lower at 0.5 mm and 1.0 mm from the center of the fovea compared to normal eyes (P<0.001 and 0.021, respectively). At 0.5 mm and 1.0 mm from the center of the fovea, a decreased number of cones had 6 neighbors in eyes with RP (P = 0.002 for both). Greater decrease in cone density was related to disruption of the photoreceptor inner segment (IS) ellipsoid band on SD-OCT images (P = 0.044); however, dark regions were seen on AO-SLO even in areas of continuous IS ellipsoid on SD-OCT. Decreased cone density correlated thinner outer nuclear layer (P = 0.029) and thinner inner segment and outer segment thickness (P = 0.011) on SD-OCT.
Conclusions
Cone density is decreased and the regularity of the cone mosaic spatial arrangement is disrupted in eyes with RP, even when visual acuity and foveal sensitivity are good. AO-SLO imaging is a sensitive quantitative tool for detecting photoreceptor abnormalities in eyes with RP.
doi:10.1371/journal.pone.0079447
PMCID: PMC3834127  PMID: 24260224
17.  Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization 
Purpose.
To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina.
Methods.
Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc.
Results.
We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area).
Conclusions.
We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease.
We provide the first high-resolution images of retinal pericytes in the living animal. Adaptive optics ophthalmoscopy provided subcellular resolution allowing for identification of fluorescent NG2-positive cells on capillaries, venules, and arterioles in vivo. Ex vivo imaging confirmed in vivo data.
doi:10.1167/iovs.13-12581
PMCID: PMC3869420  PMID: 24150762
diabetic retinopathy; neurovascular coupling; adaptive optics; capillaries; microvascular network
18.  Temporal changes of human cone photoreceptors observed in vivo with SLO/OCT 
Biomedical Optics Express  2010;2(1):100-112.
In this study we use our previously introduced scanning laser ophthalmoscope (SLO) / transverse scanning optical coherence tomography (TS-OCT) instrument to investigate long term changes in cone photoreceptors. The instrument is capable to provide 3D information of the human cone photoreceptors with negligible eye motion artifacts due to an implemented 3D motion correction on a cellular level. This allows for an in vivo investigation of exactly the same location on the retina with cellular resolution over several days. Temporal changes in the backscattered intensity are observed and quantified within the junction between inner and outer segments of cone photoreceptors, the cone outer segments, the end tips of cone photoreceptors and the retinal pigment epithelium. Furthermore, the length of individual cone outer segments is measured and observed over time. We show, to the best of our knowledge for the first time, that bright reflection spots which are located within the outer segment of cone photoreceptors change their position when observed over extended time periods. The average measured bright reflection spot motion speed corresponds well to the expected cone growth speed. We believe that this observation can be associated with the first direct in vivo imaging of the cone renewal process.
doi:10.1364/BOE.2.000100
PMCID: PMC3028485  PMID: 21326640
(170.4500) Optical coherence tomography; (330.5310) Vision – photoreceptors; (170.5755) Retina scanning; (170.4470) Ophthalmology; (170.2655) Functional monitoring and imaging
19.  Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish 
PLoS ONE  2013;8(11):e80483.
Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.
doi:10.1371/journal.pone.0080483
PMCID: PMC3841302  PMID: 24303018
20.  Changes in Cellular Structures Revealed by Ultra-high Resolution Retinal Imaging in Optic Neuropathies 
PURPOSE
To study the integrity of inner and outer retinal layers in patients with various types of optic neuropathy by using high-resolution imaging modalities.
METHODS
Three high-resolution imaging systems constructed at the University of California Davis were used to acquire retinal images from patients with optic neuropathy: (1) adaptive optics (AO)-flood–illuminated fundus camera, (2) high-resolution Fourier domain optical coherence tomography (FDOCT), and (3) adaptive optics-Fourier domain optical coherence tomography (AO-FDOCT). The AO fundus camera provides en face images of photoreceptors whereas cross-sectional images (B-scans) of the retina are obtained with both FDOCT and AO-FDOCT. From the volumetric FDOCT data sets, detailed thickness maps of a three-layer complex consisting of the nerve fiber (NF), ganglion cell (GC), and inner plexiform (IP) layers were created. The number of visible cones in the en face images of photoreceptors was then compared with visual sensitivity maps from Humphrey visual field (HVF; Carl Zeiss Meditec, Inc., Dublin, CA) testing, as well as FDOCT and AO-FDOCT images, including the thickness maps of the NF–GC–IP layer complex. Five types of optic neuropathy were studied: (1) optic neuritis with multiple sclerosis (MS), (2) idiopathic intracranial hypertension (pseudotumor cerebri), (3) nonarteritic anterior ischemic optic neuropathy (NAION), (4) optic nerve head drusen with NAION, and (5) systemic lupus erythematosus with MS and arthritis.
RESULTS
With permanent visual field loss and thinning of the NF–GC–IP layer complex, cone photoreceptors showed structural changes, making them less reflective, which caused the appearance of dark spaces in the en face images (hence, reduced number of visible cones) and indistinct outer retinal layers in OCT images. However, when the visual field loss was only transient, with a normal NF–GC–IP layer complex, there were no detectable abnormalities in cone photoreceptors (i.e., they were densely packed and had distinct photoreceptor layering in the OCT images).
CONCLUSIONS
Cone photoreceptors show structural changes when there is permanent damage to overlying inner retinal layers. There was a positive relation between the thickness of the three-layer inner retinal complex, visual sensitivity, and integrity of the cone mosaic.
doi:10.1167/iovs.07-0980
PMCID: PMC2707837  PMID: 18436843
21.  Integrity of the Cone Photoreceptor Mosaic in Oligocone Trichromacy 
Oligocone trichromacy (OT) is an unusual cone dysfunction syndrome associated with normal or near-normal color vision. In this paper, the authors describe novel observations on the underlying structural basis of OT at the level of the cone mosaic.
Purpose.
Oligocone trichromacy (OT) is an unusual cone dysfunction syndrome characterized by reduced visual acuity, mild photophobia, reduced amplitude of the cone electroretinogram with normal rod responses, normal fundus appearance, and normal or near-normal color vision. It has been proposed that these patients have a reduced number of normal functioning cones (oligocone). This paper has sought to evaluate the integrity of the cone photoreceptor mosaic in four patients previously described as having OT.
Methods.
Retinal images were obtained from two brothers (13 and 15 years) and two unrelated subjects, one male (47 years) and one female (24 years). High-resolution images of the cone mosaic were obtained using high-speed adaptive optics (AO) fundus cameras. Visible structures were analyzed for density using custom software. Additional retinal images were obtained using spectral domain optical coherence tomography (SD-OCT), and the four layers of the photoreceptor-retinal pigment epithelium complex (ELM, IS/OS, RPE1, RPE2) were evaluated. Cone photoreceptor length and the thickness of intraretinal layers were measured and compared to previously published normative data.
Results.
The adult male subject had infantile onset nystagmus while the three other patients did not. In the adult male patient, a normal appearing cone mosaic was observed. However, the three other subjects had a sparse mosaic of cones remaining at the fovea, with no structure visible outside the central fovea. On SD-OCT, the adult male subject had a very shallow foveal pit, with all major retinal layers being visible, and both inner segment (IS) and outer segment (OS) length were within normal limits. In the other three patients, while all four layers were visible in the central fovea and IS length was within normal limits, the OS length was significantly decreased. Peripherally the IS/OS layer decreased in intensity, and the RPE1 layer was no longer discernable, in keeping with the lack of cone structure observed on AO imaging outside the central fovea.
Conclusions.
Findings are consistent with the visual deficits being caused by a reduced number of healthy cones in the two brothers and the adult female. In the unrelated adult subject, no structural basis for the disorder was found. These data suggest two distinct groups on the basis of structural imaging. It is proposed that the former group with evidence of a reduction in cone numbers is more in keeping with typical OT, with the latter group representing an OT-like phenotype. These two groups may be difficult to readily discern on the basis of phenotypic features alone, and high-resolution imaging may be an effective way to distinguish between these phenotypes.
doi:10.1167/iovs.10-6659
PMCID: PMC3175968  PMID: 21436275
22.  Phenotypic Characteristics Including In Vivo Cone Photoreceptor Mosaic in KCNV2-Related “Cone Dystrophy with Supernormal Rod Electroretinogram” 
Investigative ophthalmology & visual science  2013;54(1):10.1167/iovs.12-10971.
Purpose
To report phenotypic characteristics including macular cone photoreceptor morphology in KCNV2-related “cone dystrophy with supernormal rod electroretinogram” (CDSR).
Methods
Seven patients, aged 9 to 18 years at last visit, with characteristic full-field electroretinographic (ERG) features of CDSR were screened for mutations in the KCNV2 gene. All patients underwent detailed ophthalmological evaluation, which included distance and color vision testing, contrast sensitivity measurement, fundus photography, fundus auto-fluorescence (FAF) imaging, and spectral domain-optical coherence tomography (SD-OCT). Follow-up visits were available in six cases. Rod photoreceptor function was assessed using a bright white flash ERG protocol (240 cd·s/m2). Macular cone photoreceptor morphology was assessed from 2° by 2° zonal images obtained using adaptive optics scanning laser ophthalmoscopy (AOSLO) in six cases.
Results
Pathogenic mutations in KCNV2 were identified in all seven cases. Best corrected vision was 20/125 or worse in all cases at the latest visit (20/125–20/400). Vision loss was progressive in two cases. Color vision and contrast sensitivity was abnormal in all cases. Retinal exam revealed minimal pigment epithelial changes at the fovea in four cases. A peri- or parafoveal ring of hyperfluorescence was the most common FAF abnormality noted (five cases). The SD-OCT showed outer retinal abnormalities in all cases. The rod photoreceptor maximal response was reduced but rod sensitivity was normal. AOSLO showed markedly reduced cone density in all six patients tested.
Conclusions
Central vision parameters progressively worsen in CDSR. Structural retinal and lipofuscin accumulation abnormalities are commonly present. Macular cone photoreceptor mosaic is markedly disrupted early in the disease.
doi:10.1167/iovs.12-10971
PMCID: PMC3880354  PMID: 23221069 CAMSID: cams3789
23.  Noninvasive imaging of the human rod photoreceptor mosaic using a confocal adaptive optics scanning ophthalmoscope 
Biomedical Optics Express  2011;2(7):1864-1876.
The rod photoreceptors are implicated in a number of devastating retinal diseases. However, routine imaging of these cells has remained elusive, even with the advent of adaptive optics imaging. Here, we present the first in vivo images of the contiguous rod photoreceptor mosaic in nine healthy human subjects. The images were collected with three different confocal adaptive optics scanning ophthalmoscopes at two different institutions, using 680 and 775 nm superluminescent diodes for illumination. Estimates of photoreceptor density and rod:cone ratios in the 5°–15° retinal eccentricity range are consistent with histological findings, confirming our ability to resolve the rod mosaic by averaging multiple registered images, without the need for additional image processing. In one subject, we were able to identify the emergence of the first rods at approximately 190 μm from the foveal center, in agreement with previous histological studies. The rod and cone photoreceptor mosaics appear in focus at different retinal depths, with the rod mosaic best focus (i.e., brightest and sharpest) being at least 10 μm shallower than the cones at retinal eccentricities larger than 8°. This study represents an important step in bringing high-resolution imaging to bear on the study of rod disorders.
doi:10.1364/BOE.2.001864
PMCID: PMC3130574  PMID: 21750765
(110.1080) Active or adaptive optics; (330.5310) Vision; photoreceptors; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
24.  Analysis of macular cone photoreceptors in a case of occult macular dystrophy 
Purpose
To investigate changes in cone photoreceptors with adaptive optics (AO) fundus imaging and spectral domain optical coherence tomography (SD-OCT) in a case of occult macular dystrophy (OMD).
Patient and methods
Both eyes of a 42-year-old woman diagnosed with OMD were examined. We used an AO fundus camera to obtain images of cone photoreceptors in the macula of the OMD subject and five healthy control subjects. Correlations between the AO images and the SD-OCT images were examined. Cone photoreceptors in eight areas in the macula of OMD and healthy control subjects were analyzed and compared.
Results
SD-OCT showed a loss of the cone outer-segment tips line outside of the fovea in both eyes of the subject with OMD. The left eye with decreased visual acuity showed a discontinuous photoreceptor inner-segment and outer-segment line and cone outer-segment tips line at the fovea in SD-OCT and loss of cone mosaics as a dark spot in the AO image. In panoramic AO images and cone-density maps, less cone density was observed in a ring-like region outside the fovea than in the peripheral retina. In most of the areas examined, the cone densities were lower in the OMD eyes than in the healthy control eyes.
Conclusions
Cone densities in the macula of the OMD patient were greatly decreased. AO images were found to be useful to evaluate morphologic changes in cone photoreceptors in patients with OMD.
doi:10.2147/OPTH.S44446
PMCID: PMC3659265  PMID: 23696695
occult macular dystrophy; adaptive optics; cone photoreceptor; cone analysis; optical coherence tomography
25.  In vivo imaging of the photoreceptor mosaic of a rod monochromat 
Vision research  2008;48(26):2564-2568.
Complete achromatopsia (i.e., rod monochromacy) is a congenital vision disorder in which cone function is absent or severely diminished, often due to mutations in one of two components of the cone photo-transduction cascade (transducin or the cyclic-nucleotide gated channel). Previous histological data concerning cone structure are conflicting; suggesting anywhere from normal numbers of foveal cones to a complete absence of foveal cones. Here, we used an adaptive optics ophthalmoscope to obtain in vivo retinal images from a rod monochromat for whom the genetic basis of the disorder consists of a homozygous mutation in the CNGB3 gene. Behavioral data from the patient were consistent with an absence of cone function. Retinal images revealed a severely disrupted photoreceptor mosaic in the fovea and parafovea, where the size and density of the visible photoreceptors resembled that of normal rods. Imaging of additional rod monochromats to characterize differences in the photoreceptor mosaic between genetically classified patients will be required to determine which, if any, might be receptive to restorative gene therapy procedures.
doi:10.1016/j.visres.2008.04.006
PMCID: PMC2582057  PMID: 18499214
Cones; Rods; Adaptive optics; Achromatopsia; Retinal disease; Waveguiding

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