Visual Pigment Research Articles

Straying from the flatfish retinal plan: Cone photoreceptor patterning in the common sole (Solea solea) and the Senegalese sole (Solea senegalensis).

The retinas of nonmammalian vertebrates have cone photoreceptor mosaics that are often organized as highly patterned lattice-like distributions. In fishes, the two main lattice-like patterns are composed of double cones and single cones that are either assembled as interdigitized squares or as alternating rows. The functional significance of such orderly patterning is unknown. Here, the cone mosaics in two species of Soleidae flatfishes, the common sole and the Senegalese sole, were characterized and compared to those from other fishes to explore variability in cone patterning and how it may relate to visual function. The cone mosaics of the common sole and the Senegalese sole consisted of single, double, and triple cones in formations that differed from the traditional square mosaic pattern reported for other flatfishes in that no evidence of higher order periodicity was present. Furthermore, mean regularity indices for single and double cones were conspicuously lower than those of other fishes with "typical" square and row mosaics, but comparable to those of goldfish, a species with lattice-like periodicity in its cone mosaic. Opsin transcripts detected by quantitative polymerase chain reaction (sws1, sws2, rh2.3, rh2.4, lws, and rh1) were uniformly expressed across the retina of the common sole but, in the Senegalese sole, sws2, rh2.4, and rh1 were more prevalent in the dorsal retina. Microspectrophotometry revealed five visual pigments in the retina of the common sole [S(472), M(523), M(536), L(559), and rod(511)] corresponding to the repertoire of transcripts quantified except for sws1. Overall, these results indicate a loss of cone mosaic patterning in species that are primarily nocturnal or dwell in low light environments as is the case for the common sole and the Senegalese sole. The corollary is that lattice-like patterning of the cone mosaic may improve visual acuity. Ecological and physiological correlates derived from observations across multiple fish taxa that live in low light environments and do not possess lattice-like cone mosaics are congruent with this claim.

Color discrimination assessment in patients with hypothyroidism using the Farnsworth-Munsell 100 hue test.

There is evidence in the literature that hypofunction of the thyroid gland (hypothyroidism) affects color vision in rodents by influencing the production of the visual pigment opsin. The effect of hypothyroidism on color vision in humans has not been examined in any great detail. In this cross-sectional study we evaluated color discrimination using the Farnsworth-Munsell 100 hue test (FM-100 test) in 25 individuals with pre-treatment hypothyroidism (mean age ${38}\;{\pm }\;{9}.{2}\;\text{yr}$38±9.2yr), and a control euthyroid group, ${ n} ={26}$n=26 (mean age ${39.6}\;{\pm }\;{8}.{4}\;\text{yr}$39.6±8.4yr). There was no statistically significant difference in the total error score ($\surd{\text{TES}}$√TES) between the groups, but the hypothyroid group had a significantly greater partial error score ($\surd{\text{PES}}$√PES) along the blue-yellow (B-Y) axis compared to the red-green (R-G) axis. No statistically significant differences in B-Y and R-G PES were observed in the control group. This study shows that hypothyroidism affects color vision in humans, causing significant impairment in the B-Y color subsystem.

Vitamin A: its many roles-from vision and synaptic plasticity to infant mortality.

The recognition that a dietary factor is essential to maintain good and sensitive vision as well as overall health goes back over 3,000years to the ancient Egyptians. With the discovery of the vitamins at the turn of the twentieth century, fat-soluble vitamin A was soon shown to be the essential factor. In the first half of the twentieth century, the role vitamin A plays in vision, as precursor to the light-sensitive visual pigment molecules in the photoreceptors was elegantly worked out, especially by George Wald and his colleagues. Beginning in the 1960s, with the recognition of the active metabolite of vitamin A, its acid form now called retinoic acid, the roles of vitamin A in maintaining overall health of an organism began to be explored, and this research continues to this day. Receptors activated by retinoic acid, the RARs and RXRs have been shown to regulate gene transcription in a surprisingly wide variety of biological processes from early growth and development to the maintenance of epithelial tissues in many organs, the regulation of the immune system, and even the modulation of synaptic function in the brain involved in mechanisms underlying memory and learning. Therapeutic uses for retinoic acid have been developed, including one for a specific form of leukemia. The story is by no means complete and it is likely more surprises await with regard to this remarkable molecule.

Extending Human Vision to Infrared and Ultraviolet Light: A Study Using Micro-Particles and Fluorescent Molecules

In this study, rare earth doped infrared (IR) to visible, up-converting particles along-with efficient ultraviolet (UV) to visible fluorescent molecules were imbedded in proteins and were used as a mean for increasing the human vision range to infrared and ultraviolet wavelengths. Stilbene-420 fluorescent molecules which convert near UV light to blue light, were chosen for the strong overlap of their fluorescence emission with the sensitivity region of blue cone cells and for the ability of blue light to increase the regeneration of bleached visual pigments. Our data show that the up-conversion efficiency of the IR up-converting particles and blue fluorescing molecules efficiencies remained unchanged when these material are imbedded in proteins compared to their efficiencies in water solutions. Addition of up-converting particles to rod visual cells resulted in the bleaching of the visual pigments, rods, when irradiated with infrared light (980 nm) whereas no bleaching was observed, under the same conditions, without the presence of up-converting particles. This suggests that the up-converted green light induced visual process in the rod visual pigments. In addition, we describe the design and present data of a novel optical device, which can be used as eye glasses, utilizing up-converting particles that allows the wearer to see rather intense infrared light.

Photoreceptors and diurnal variation in spectral sensitivity in the fiddler crab Gelasimus dampieri.

Colour signals, and the ability to detect them, are important for many animals and can be vital to their survival and fitness. Fiddler crabs use colour information to detect and recognise conspecifics, but their colour vision capabilities remain unclear. Many studies have attempted to measure their spectral sensitivity and identify contributing retinular cells, but the existing evidence is inconclusive. We used electroretinogram (ERG) measurements and intracellular recordings from retinular cells to estimate the spectral sensitivity of Gelasimus dampieri and to track diurnal changes in spectral sensitivity. G. dampieri has a broad spectral sensitivity and is most sensitive to wavelengths between 420 and 460 nm. Selective adaptation experiments uncovered an ultraviolet (UV) retinular cell with a peak sensitivity shorter than 360 nm. The species' spectral sensitivity above 400 nm is too broad to be fitted by a single visual pigment and using optical modelling, we provide evidence that at least two medium-wavelength sensitive (MWS) visual pigments are contained within a second blue-green sensitive retinular cell. We also found a ∼25 nm diurnal shift in spectral sensitivity towards longer wavelengths in the evening in both ERG and intracellular recordings. Whether the shift is caused by screening pigment migration or changes in opsin expression remains unclear, but the observation shows the diel dynamism of colour vision in this species. Together, these findings support the notion that G. dampieri possesses the minimum requirement for colour vision, with UV and blue/green receptors, and help to explain some of the inconsistent results of previous research.

Visual cells and visual pigments of the river lamprey revisited.

Retinas of the river lamprey Lampetra fluviatilis were studied by microspectrophotometry, electroretinography and single-photoreceptor electrophysiology to reconcile the apparently contradictory conclusions on the nature of lamprey photoreceptor cells drawn in the early work by Govardovskii and Lychakov (J Comp Physiology A 154:279-286, 1984) and in recent studies. In agreement with recent works, we confirmed former identification of short photoreceptors as rods and of long photoreceptors as cones. In line with the results of 1984, we show that within a certain range of light intensities the lamprey retina exhibits "color discrimination". We found that the overlap of working intensity ranges of rods and cones is not a unique feature of lamprey short receptors, and suggest that rod-cone (possibly color) vision may be common among vertebrates. We show that the decay of meta-intermediates in lamprey cones occurs almost 100 times faster than in typical rod metarhodopsins. Rate of decay of metarhodopsins of lamprey rods take an intermediate position between typical rods and cones. This makes lamprey rhodopsin similar to transmuted cone visual pigment in "rods" of nocturnal geckos. We argue that defining various types of photoreceptors as simply "rods" and "cones" may be functionally correct, but neglects their genetic, biochemical and morphological features and evolutionary history.

Atomic force microscopy in the study of retinal structure

The review addresses the current state of atomic force microscopy (AFM) usage in ophthalmology. Briefly presented here are the history of the development of AFM, principles and modes of operation, its advantages, disadvantages, as well as a comparison with other types of microscopy. The review describes the capabilities of AFM in visualization of various structures of the eye. A significant part of the review is devoted to the study of the retina, which arouses great interest among researchers. In particular, the possibilities of AFM for visualization at the submicron level of various structures in the retina, such as the internal limiting membrane, membrane cells, Müller cells, retinal pigment epithelium in their normal state and in the presence of a pathology (age-related macular degeneration, diabetes mellitus) were reviewed. In addition, several study papers were analyzed, providing a base for the judgement of the mechanical properties of said structures. An AFM study of the visual pigment rhodopsin helped identify its dimeric structure. The stability of the rhodopsin molecule was proved to be determined by the degree of strength of its individual segments connected to each other. The AFM method is a highly accurate method that helps solve many fundamental and practical problems, particularly in ophthalmology.

Разнообразие и адаптивные свойства зрительных пигментов рыб с изменяемой окраской роговицы

The properties of the visual pigments in several fish species that possess a unique physiological mechanism of changing the corneal colouration depending on the intensity of ambient illumination are described in the present paper. The absorbance spectra of the visual pigments and the spectral characteristics of the cornea have been measured in masked greenling Hexagrammos octogrammus and five fish species of the order Tetraodontiformes using microspectrophotometry. In all the species, retinal photoreceptors contain a mixture of the rhodopsin (A1) and porphyropsin (A2) pigments in different proportions, from 0 to 100%. The experiments with double cones in a chloride-free medium have revealed a shift of λmax in some of the pigments to the shortwave region (ionochromic effect). This suggests that pufferfishes have one pigment of the LWS type (λmax = A2_567 nm), while greenling has two LWS-pigments (λmax = A2_625, and A1_560 nm). The shift of the spectral absorbance of double cones towards the longwave region in pufferfish, as compared to that in most other shallow-sea fishes, is probably explained by the presence of a changeable yellow-orange carotenoid corneal filter.

Phosphorylation at Serine 21 in G protein-coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors.

Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.

Mimicking light-sensing chromophore in visual pigments and determination isomerization site

Three retinal derivatives are designed and synthesized under the inspiration of natural visual pigments. The retinal derivative V3 (retinal-phenylenediamine) is able to respond sensitively to visual light in the absence of a protein environment through isomerization and deprotonation. The response process is applied to verification of information security.

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Visual Opsin Diversity in Sharks and Rays.

The diversity of color vision systems found in extant vertebrates suggests that different evolutionary selection pressures have driven specializations in photoreceptor complement and visual pigment spectral tuning appropriate for an animal's behavior, habitat, and life history. Aquatic vertebrates in particular show high variability in chromatic vision and have become important models for understanding the role of color vision in prey detection, predator avoidance, and social interactions. In this study, we examined the capacity for chromatic vision in elasmobranch fishes, a group that have received relatively little attention to date. We used microspectrophotometry to measure the spectral absorbance of the visual pigments in the outer segments of individual photoreceptors from several ray and shark species, and we sequenced the opsin mRNAs obtained from the retinas of the same species, as well as from additional elasmobranch species. We reveal the phylogenetically widespread occurrence of dichromatic color vision in rays based on two cone opsins, RH2 and LWS. We also confirm that all shark species studied to date appear to be cone monochromats but report that in different species the single cone opsin may be of either the LWS or the RH2 class. From this, we infer that cone monochromacy in sharks has evolved independently on multiple occasions. Together with earlier discoveries in secondarily aquatic marine mammals, this suggests that cone-based color vision may be of little use for large marine predators, such as sharks, pinnipeds, and cetaceans.

A detailed investigation of the visual system and visual ecology of the Barrier Reef anemonefish, Amphiprion akindynos

Vision plays a major role in the life of most teleosts, and is assumingly well adapted to each species ecology and behaviour. Using a multidisciplinary approach, we scrutinised several aspects of the visual system and ecology of the Great Barrier Reef anemonefish, Amphiprion akindynos, including its orange with white patterning, retinal anatomy and molecular biology, its symbiosis with anemones and sequential hermaphroditism. Amphiprion akindynos possesses spectrally distinct visual pigments and opsins: one rod opsin, RH1 (498 nm), and five cone opsins, SWS1 (370 nm), SWS2B (408 nm), RH2B (498 nm), RH2A (520 nm), and LWS (554 nm). Cones were arranged in a regular mosaic with each single cone surrounded by four double cones. Double cones mainly expressed RH2B (53%) in one member and RH2A (46%) in the other, matching the prevailing light. Single cones expressed SWS1 (89%), which may serve to detect zooplankton, conspecifics and the host anemone. Moreover, a segregated small fraction of single cones coexpressed SWS1 with SWS2B (11%). This novel visual specialisation falls within the region of highest acuity and is suggested to increase the chromatic contrast of Amphiprion akindynos colour patterns, which might improve detection of conspecifics.

LWS visual pigment in owls: Spectral tuning inferred by genetics

Owls constitute a diverse group of raptors, active at different times of the day with distinct light conditions that might be associated with multiple visual adaptations. We investigated whether shifts in the spectral sensitivity of the L cone visual pigment, as inferred by analysis of gene structure, could be one such adaptive mechanism. Using Sanger sequencing approach, we characterized the long wavelength-sensitive (LWS) opsin gene expressed in the retina of five owl species, specifically chosen to represent distinct patterns of activity. Nocturnality was epitomized by the American barn owl (Tyto furcata), the striped owl (Asio clamator), and the tropical screech owl (Megascops choliba); diurnality, by the ferruginous pygmy owl (Glaucudium brasilianum); and cathemerality, by the burrowing owl (Athene cunicularia). We also analyzed the presence of the L cone in the retinas of four species of owl (T. furcata, A. cunicularia, G. brasilianum and M. choliba) using immnunohistochemistry. Five critical sites for the spectral tuning of the LWS opsin (164, 181, 261, 269, and 292) were analyzed and compared to the sequence of other birds. The sequence of A. cunicularia showed a substitution on residue 269, with the presence of an alanine instead threonine, which generates an estimated maximum absorption (λmax) around 537 nm. No other variation was found in the spectral tuning sites of the LWS opsin, among the other species, and the λmax was estimated at around 555 nm. The presence of L cones in the retinas of the four species of owls was revealed using immunohistochemistry and we observed a reduced number of L cones in T. furcata compared to A. cunicularia, G. brasilianum and M. choliba.

Growth hormone regulates opsin expression in the retina of a salmonid fish.

Colour vision relies on retinal photoreceptors that express a different predominant visual pigment protein (opsin). In several vertebrates, the primary opsin expressed by a photoreceptor can change throughout ontogeny, although the molecular factors that influence such regulation are poorly understood. One of these factors is thyroid hormone which, together with its receptors, modulates opsin expression in the retinas of multiple vertebrates including rodents and salmonid fishes. In the latter, thyroid hormone induces a switch in opsin expression from SWS1 (ultraviolet light sensitive) to SWS2 (short wavelength or blue light sensitive) in the single cone photoreceptors of the retina. The actions of other hormones on opsin expression have not been investigated. In the present study, we used a transgenic strain of coho salmon (Oncorhynchus kitsutch) with enhanced levels of circulating growth hormone compared to that of wild siblings to assess the effects of this hormone on the SWS1 to SWS2 opsin switch. Transgenic fish showed a developmentally accelerated opsin switch compared to size-matched controls as assessed by immunohistological and in situ hybridisation labelling of photoreceptors and by quantification of transcripts using quantitative polymerase chain reaction. This accelerated switch led to a different spectral sensitivity maximum, under a middle to long wavelength adapting background, from ultraviolet (λmax ~380nm) in controls to short wavelengths (λmax ~430nm) in transgenics, demonstrating altered colour vision. The effects of growth hormone over-expression were independent of circulating levels of thyroid hormone (triiodothyronine), the hormone typically associated with opsin switches in vertebrates.

Transcriptomic data support a nocturnal bottleneck in the ancestor of gecko lizards

Gecko lizards are a species-rich clade of primarily-nocturnal squamate reptiles. In geckos, adaptations to nocturnality have dramatically reshaped the eye. Perhaps the most notable change is the loss of rod cells in the retina and subsequent “transmutation” of cones into a rod-like morphology and physiology. While many studies have noted the absence of some rod-specific genes, such as the visual pigment Rhodopsin (RH1), these studies have focused on just a handful of species that are nested deep in the gecko phylogeny. Thus, it is not clear whether these changes arose through convergence, are homologous and ubiquitous across geckos, or restricted to a subset of species. Here, we used de novo eye transcriptomes from five gecko species, and genomes from two additional gecko species, representing the breadth of extant gecko diversity (i.e. 4 of the 7 gecko families, spanning the deepest divergence of crown Gekkota), to show that geckos lost expression of almost the entire suite of necessary rod-cell phototransduction genes in the eye, distinct from all other squamate reptiles. Geckos are the first vertebrate group to have lost their complete rod-cell expression pathway, not just the visual pigment. In addition, all sampled species have also lost expression of the cone-opsin SWS2 visual pigment. These results strongly suggest a single loss of rod cells and subsequent cone-to-rod transmutation that occurred prior to the diversification of extant geckos.

Unusual A1/A2–visual pigment conversion during light/dark adaptation in marine fish

Changes in visual pigments were studied in two marine fish species, the masked greenling Hexagrammos octogrammus and the prickleback Pholidapus dybowskii. A microspectrophotometric (MSP) analysis showed that the rods and cones of the fish collected from the natural marine environment in summer or kept in a tank at a high illumination level predominantly contained porphyropsins based on chromophore A2. As a result, λmax of the double cones significantly shifted to longer wavelengths, reaching 625 and 609 nm, respectively. After several weeks of dark adaptation, the spectra of all the photoreceptor types shifted to shorter wavelengths, as the A1:A2 ratio switched to A1. The MSP data from the fish kept under controlled light conditions were confirmed by chromatography (HPLC), which showed that the changes in the chromophore ratio were reversible and independent of the water temperature. After the preliminary deep dark adaptation, the first noticeable shift in the pigment ratio from A1 to A2 occurred within two weeks of exposure to bright light. A novel finding in this study was a reverse polarity of A1/A2 changes, unlike the case in most other fish species, where A2 chromophore predominated after the dark exposure. This demonstration of the unusual phenomenon of visual pigment transformation suggests a modification or a new way for the activation of specific biochemical mechanisms of A1:A2 conversion at both high and low illumination levels.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Two-photon microperimetry: sensitivity of human photoreceptors to infrared light.

Microperimetry is a subjective ophthalmologic test used to assess retinal function at various specific and focal locations of the visual field. Historically, visible light has been described as ranging from 400 to 720 nm. However, we previously demonstrated that infra-red light can initiate visual transduction in rod photoreceptors by a mechanism of two-photon absorption by visual pigments. Here we introduce a newly designed and constructed two-photon microperimeter. We provide for the first time evidence of the presence of a nonlinear process occurring in the human retina based on psychophysical tests using newly developed instrumentation. Since infra-red light penetrates the aged front of the eye better than visible light, it has the potential for improved functional diagnostics in patients with age-related visual disorders.

Sensitivity of Mammalian Cone Photoreceptors to Infrared Light

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism. To investigate selectively mammalian cone signaling in mice, we used animals with disabled rod signal transduction. We found that, contrary to the expected progressive sensitivity decrease based on the one-photon cone visual pigment spectral template, the sensitivity of mouse cone photoreceptors decreases only up to 800 nm and then increases at 900 nm and 1000 nm. Similarly, in experiments with the parafoveal region of macaque retinas, we found that the spectral sensitivity of primate cones diverged above the predicted one-photon spectral sensitivity template beyond 800 nm. In both cases, efficient detection of IR light was dependent on minimizing the dispersion of the ultrashort light pulses, indicating a non-linear two-photon activation process. Together, our studies demonstrate that mammalian cones can be activated by near IR light by a nonlinear two-photon excitation. Our results pave the way for the creation of a two-photon IR-based ophthalmoscope for the simultaneous imaging and functional testing of human retinas as a novel tool for the diagnosis and treatment of a wide range of visual disorders.