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.
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.
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.
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.
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.
(330.4460) Ophthalmic optics and devices; (180.4315) Nonlinear microscopy; (170.0110) Imaging systems
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.
retina; adaptive optics; imaging, photoreceptors; retinal pigment epithelium; retinal ganglion cells
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.
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.
(110.1080) Active or adaptive optics; (220.1230) Apodization; (170.3880) Medical and biological imaging; (170.4460) Ophthalmic optics and devices
This is the first in vivo study of cone photoreceptor packing density at different ages.
To study the variation of cone photoreceptor packing density across the retina in healthy subjects of different ages.
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.
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.
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.
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.
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.
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.
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.
We have used an adaptive-optics scanning laser ophthalmoscope to image the cone photoreceptor mosaic throughout the central 10°–12° of the retina for four normal subjects. We then constructed montages of the images and processed the montages to determine cone locations. Cone densities range from approximately 10,000 cones/mm2 at 7° to 40,000 cones/mm2 at 1°. The smallest cones were not resolved in the center of the fovea. From the locations of the cones we also analyzed the packing properties of the cone mosaic, finding that all four subjects had a slight cone streak of increased cone density and that, in agreement with previous studies using different approaches, the packing geometry decreased in regularity from the fovea toward the periphery. We also found variations in packing density between subjects and in local anisotropy across retinal locations. The complete montages are presented for download, as well as the estimated cone locations.
To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations.
Eleven subjects were recruited, eight of whom have been previously described. Cone and rod density was measured using images of the photoreceptor mosaic obtained from an adaptive optics scanning light ophthalmoscope (AOSLO). Total retinal thickness, inner retinal thickness, and outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness were measured using cross-sectional spectral-domain optical coherence tomography (SD-OCT) images. Molecular genetic analyses were performed to characterize the OPN1LW/OPN1MW gene array.
While disruptions in retinal lamination and cone mosaic structure were observed in all subjects, genotype-specific differences were also observed. For example, subjects with “L/M interchange” mutations resulting from intermixing of ancestral OPN1LW and OPN1MW genes had significant residual cone structure in the parafovea (∼25% of normal), despite widespread retinal disruption that included a large foveal lesion and thinning of the parafoveal inner retina. These subjects also reported a later-onset, progressive loss of visual function. In contrast, subjects with the C203R missense mutation presented with congenital blue cone monochromacy, with retinal lamination defects being restricted to the ONL+HFL and the degree of residual cone structure (8% of normal) being consistent with that expected for the S-cone submosaic.
The photoreceptor phenotype associated with OPN1LW and OPN1MW mutations is highly variable. These findings have implications for the potential restoration of visual function in subjects with opsin mutations. Our study highlights the importance of high-resolution phenotyping to characterize cellular structure in inherited retinal disease; such information will be critical for selecting patients most likely to respond to therapeutic intervention and for establishing a baseline for evaluating treatment efficacy.
Subjects with OPN1LW and OPN1MW mutations showed a spectrum of retinal phenotypes with genotype-specific differences. This has implications for restoration of visual function in these subjects and highlights high-resolution retinal imaging as a complementary tool for emerging therapeutic efforts.
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.
Cones; Rods; Adaptive optics; Achromatopsia; Retinal disease; Waveguiding
Using adaptive optics imaging tools to image the living retina, numerous investigators have reported temporal fluctuation in the reflectivity of individual cone photoreceptors. In addition, there is cone-to-cone (spatial) variation in reflectivity. As it has only recently become possible to image the complete rod photoreceptor mosaic in the living human retina, we sought to characterize the reflectivity of individual rods and compare their behavior to that of foveal/parafoveal cones. Across two subjects, we were able to successfully track the reflectance behavior of 1,690 rods and 1,980 cones over 12 hours. Rod and cone photoreceptors showed similar regional and temporal variability in their reflectance profiles, suggesting the presence of a common governing physiological process. Within the rod and cone mosaics, there was no sign of spatial clumping of reflectance profile behavior; that is, the arrangement of cells of a given archetypal reflectance profile within the mosaic was indistinguishable from random. These data demonstrate the ability to track the behavior of rod reflectivity over time. Finally, as these and other reflectance changes may be an indicator of photoreceptor function, a future extension of this method will be to analyze this behavior in patients with rod photoreceptor dysfunction (e.g., retinitis pigmentosa, Usher’s syndrome, and congenital stationary night blindness).
(110.1080) Active or adaptive optics; (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (330.7331) Visual optics, receptor optics; (330.5310) Vision-photoreceptors
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.
(170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering; (110.1080) Active or adaptive optics
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.
(170.4500) Optical coherence tomography; (330.5310) Vision – photoreceptors; (170.5755) Retina scanning; (170.4470) Ophthalmology; (170.2655) Functional monitoring and imaging
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.
(110.1080) Active or adaptive optics; (330.5310) Vision; photoreceptors; (170.1610) Clinical applications; (170.3880) Medical and biological imaging; (170.4470) Ophthalmology
An ultrasmall spot size scanning laser ophthalmoscope has been developed that employs an annular aberration-corrected incident beam to increase the effective numerical aperture of the eye thereby reducing the width of the probing light spot. Parafovea and foveal cone photoreceptor visibility determined from small area retinal image scans are discussed from the perspective of mode matching between the focused incident beam and the waveguide modes of individual cones. The cone visibility near the fovea centralis can be increased with the annular illumination scheme whereas the visibility of larger parafovea cones drops significantly as a consequence of poorer mode match. With further improvements of the implemented wavefront correction technology it holds promise for individual cone-photoreceptor imaging at the fovea centralis and for optical targeting of the retina with increased resolution.
(170.0110) Imaging systems; (170.5755) Retina scanning; (330.5310) Vision; photoreceptors; (330.7327) Visual optics, ophthalmic instrumentation
Autofluorescence fundus imaging using an adaptive optics scanning laser ophthalmoscope (AOSLO) allows for imaging of individual retinal pigment epithelial (RPE) cells in vivo. In this study, the potential of retinal damage was investigated by using radiant exposure levels that are 2 to 150 times those used for routine imaging.
Macaque retinas were imaged in vivo with a fluorescence AOSLO. The retina was exposed to 568- or 830-nm light for 15 minutes at various intensities over a square ½° per side. Pre-and immediate postexposure images of the photoreceptors and RPE cells were taken over a 2° field. Long-term AOSLO imaging was performed intermittently from 5 to 165 days after exposure. Exposures delivered over a uniform field were also investigated.
Exposures to 568-nm light caused an immediate decrease in autofluorescence of RPE cells. Follow-up imaging revealed either full recovery of autofluorescence or long-term damage in the RPE cells at the exposure. The outcomes of AOSLO exposures and uniform field exposures of equal average power were not significantly different. No effects from 830-nm exposures were observed.
The study revealed a novel change in RPE autofluorescence induced by 568-nm light exposure. Retinal damage occurred as a direct result of total average power, independent of the light-delivery method. Because the exposures were near or below permissible levels in laser safety standards, these results suggest that caution should be used with exposure of the retina to visible light and that the safety standards should be re-evaluated for these exposure conditions.
To describe spectral-domain optical coherence tomography (SD-OCT) and adaptive optics (AO) imaging in hydroxychloroquine retinal toxicity.
Two patients with long-term hydroxychloroquine use, subtle perifoveal ophthalmoscopic pigmentary changes, and bilateral perifoveal defects on automated Humphrey visual field (HVF) 10-2 perimetry were imaged using SD-OCT and AO.
SD-OCT images demonstrated loss of photoreceptor inner segment/outer segment (IS/OS) junction and a downward “sink-hole” displacement of inner retinal structures in areas of hydroxychloroquine toxicity corresponding to HVF 10-2 defects and ophthalmoscopic clinical examination findings. SD-OCT irregularities in the IS/OS junction were also seen in areas not detected on HVF 10-2. AO images showed disruption of the cone photoreceptor mosaic in areas corresponding to HVF 10-2 defects and SD-OCT IS/OS junction abnormalities. Additionally, irregularities in the cone photoreceptor density and mosaic were seen in areas with normal HVF 10-2 and SD-OCT findings.
SD-OCT and AO detected abnormalities that correlate topographically with visual field loss from hydroxychloroquine toxicity as demonstrated by HVF 10-2 and may be useful in the detection of subclinical abnormalities that precede symptoms or objective visual field loss.
To examine retinal structure and changes in photoreceptor intensity post-dark adaptation in patients with complete congenital stationary night blindness and Oguchi disease.
Prospective observational case series.
We recruited three patients with complete congenital stationary night blindness caused by mutations in GRM6, two brothers with Oguchi disease caused by mutations in GRK1, and one normal control. Retinal thickness was measured from optical coherence tomography (OCT) images. Integrity of the rod and cone mosaic was assessed using adaptive optics scanning light ophthalmoscopy. We imaged five of the patients following a period of dark adaptation, and examined layer reflectivity on OCT in a patient with Oguchi disease under light- and dark-adapted conditions.
Retinal thickness was reduced in the parafoveal region in patients with GRM6 mutations, as a result of decreased thickness of the inner retinal layers. All patients had normal photoreceptor density at all locations analyzed. Upon removal from dark adaptation, the intensity of the rods (but not cones) in the patients with Oguchi disease gradually and significantly increased. In one Oguchi patient, the outer segment layer contrast on OCT was fourfold higher under dark-adapted versus light-adapted conditions.
The selective thinning of the inner retinal layers in patients with GRM6 mutations suggests either reduced bipolar/ganglion cell numbers or altered synaptic structure in the inner retina. Our finding that rods, but not cones, change intensity after dark adaptation suggests that fundus changes in Oguchi disease are due to changes within the rods as opposed to changes at a different retinal locus.
To ascertain the potential pathogenicity of a retinitis pigmentosa (RP)-causing RHO F45L allele in a family affected by congenital achromatopsia (ACHM).
Case series/observational study that included two patients with ACHM and 24 extended family members. Molecular genetic analysis was performed to identify RHO F45L carrier status in the family and a control population. An adaptive optics scanning light ophthalmoscope (AOSLO) was used to image the photoreceptor mosaic and assess rod and cone structure. Spectral domain optical coherence tomography (SD-OCT) was used to examine retinal lamination. Comprehensive clinical testing included acuity, color vision, and dilated fundus examination. Electroretinography was used to assess rod and cone function.
Five carriers of the RHO F45L allele alone (24–80 years) and three carriers in combination with a heterozygous CNGA3 mutant allele (10–64 years) were all free of the classic symptoms and signs of RP. In heterozygous carriers of both mutations, SD-OCT showed normal retinal thickness and intact outer retinal layers; rod and cone densities were within normal limits on AOSLO. The phenotype in two individuals affected with ACHM and harboring the RHO F45L allele was indistinguishable from that previously reported for ACHM.
The RHO F45L allele is not pathogenic in this large family; hence, the two ACHM patients would unlikely develop RP in the future.
The combined approach of comprehensive molecular analysis of individual genomes and noninvasive cellular resolution retinal imaging enhances the current repertoire of clinical diagnostic tools, giving a substantial impetus to personalized medicine.
exome sequencing; adaptive optics; rhodopsin mutations; retinitis pigmentosa; retinal degeneration
We report fast, non-scanning, wide-field two-photon fluorescence excitation with spectral and lifetime detection for in vivo biomedical applications. We determined the optical characteristics of the technique, developed a Gaussian flat-field correction method to reduce artifacts resulting from non-uniform excitation such that contrast is enhanced, and showed that it can be used for ex vivo and in vivo cellular-level imaging. Two applications were demonstrated: (i) ex vivo measurements of beta-amyloid plaques in retinas of transgenic mice, and (ii) in vivo imaging of sulfonated gallium(III) corroles injected into tumors. We demonstrate that wide-field two photon fluorescence excitation with flat-field correction provides more penetration depth as well as better contrast and axial resolution than the corresponding one-photon wide field excitation for the same dye. Importantly, when this technique is used together with spectral and fluorescence lifetime detection modules, it offers improved discrimination between fluorescence from molecules of interest and autofluorescence, with higher sensitivity and specificity for in vivo applications.
(180.4315) Microscopy; (110.4234) Imaging systems
Reflectometric techniques estimate the directionality of the retinal cones, by measuring the distribution of light at the pupil plane of light reflected off the bleached retina. The waveguide-scattering model [Marcos et al. (1998), J. Opt. Soc. Am A] predicts that the shape of this intensity distribution is determined by both the waveguide properties of the cone photoreceptors and by the topography of the cone mosaic (cone spacing). We have performed two types of cone directionality measurements. In the first type, cone directionality estimates are obtained by measuring the spatial distribution of light returning from the retina with a single entry pupil position (single-entry measurements). In the second type, estimates are obtained by measuring the total amount of light guided back through the pupil as a function of entry pupil position (multiple-entry measurements). As predicted by the model, single-entry measurements provide narrower distributions than the multiple-entry measurements, since the former are affected by both waveguides and scattering and the latter primarily by waveguides. Measurements at different retinal eccentricities and at two different wavelengths are consistent with the model. We show that the broader multiple-entry measurements are not accounted for by cone disarray. Results of multiple-entry measurements are closer to results from measurements of the psychophysical Stiles-Crawford effect (although still narrower), and the variation with retinal eccentricity and wavelength is similar. By combining single- and multiple-entry measurements we can estimate cone spacing. The estimates at 0 and 2 deg retinal eccentricities are in good agreement with published anatomical data.
To assess macular photoreceptor abnormalities in eyes with retinitis pigmentosa (RP) with preserved central vision using adaptive optics scanning laser ophthalmoscopy (AO-SLO).
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.
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.
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.
Carriers of blue cone monochromacy have fewer cone photoreceptors than normal. Here we examine how this disruption at the level of the retina affects visual function and cortical organization in these individuals. Visual resolution and contrast sensitivity was measured at the preferred retinal locus of fixation and visual resolution was tested at two eccentric locations (2.5° and 8°) with spectacle correction only. Adaptive optics corrected resolution acuity and cone spacing were simultaneously measured at several locations within the central fovea with adaptive optics scanning laser ophthalmoscopy (AOSLO). Fixation stability was assessed by extracting eye motion data from AOSLO videos. Retinotopic mapping using fMRI was carried out to estimate the area of early cortical regions, including that of the foveal confluence. Without adaptive optics correction, BCM carriers appeared to have normal visual function, with normal contrast sensitivity and visual resolution, but with AO-correction, visual resolution was significantly worse than normal. This resolution deficit is not explained by cone loss alone and is suggestive of an associated loss of retinal ganglion cells. However, despite evidence suggesting a reduction in the number of retinal ganglion cells, retinotopic mapping showed no reduction in the cortical area of the foveal confluence. These results suggest that ganglion cell density may not govern the foveal overrepresentation in the cortex. We propose that it is not the number of afferents, but rather the content of the information relayed to the cortex from the retina across the visual field that governs cortical magnification, as under normal viewing conditions this information is similar in both BCM carriers and normal controls.
Capture of light in the photoreceptor outer segment initiates a cascade of chemical events that inhibit neurotransmitter release, ultimately resulting in vision. The massed response of the photoreceptor population can be measured non-invasively by electrical recordings, but responses from individual cells cannot be measured without dissecting the retina. Here we used optical imaging to observe individual human cones in the living eye as they underwent bleaching of photopigment and associated phototransduction. The retina was simultaneously stimulated and observed with high intensity visible light at 1 kHz, using adaptive optics. There was marked variability between individual cones in both photosensitivity and pigment optical density, challenging the conventional assumption that photoreceptors act as identical subunits (coefficient of variation in rate of photoisomerization = 23%). There was also a pronounced inverse correlation between these two parameters (p<10−7); the temporal evolution of image statistics revealed this to be a dynamic relationship, with cone waveguiding efficiency beginning a dramatic increase within 3 ms of light onset. Beginning as early as 2 ms after light onset and including half of cells by ∼7 ms, cone intensity showed reversals characteristic of interference phenomena, with greater delays in reversal corresponding to cones with more photopigment (p<10−3). The timing of these changes is argued to best correspond with either the cessation of dark current, or to related events such as changes in intracellular cGMP. Cone intensity also showed fluctuations of high frequency (332±25 Hz) and low amplitude (3.0±0.85%). Other groups have shown similar fluctuations that were directly evoked by light; if this corresponds to the same phenomenon, we propose that the amplitude of fluctuation may be increased by the use of a bright flash followed by a brief pause, to allow recovery of cone circulating current.
To measure the variation in human cone photoreceptor packing density across the retina both within an individual and between individuals with different refractive errors.
A high resolution adaptive optics scanning laser ophthalmoscope was used to image the cones of eleven human eyes. Five emmetropes and six myopes were tested (+0.50D to -7.50D). For each subject we obtained four approximately 10 degree by 1.5 degree strips of cone images. Each strip started at the fovea, and proceeded towards the periphery along the four primary meridians. The position of each cone within the sampling windows was digitized manually by the investigator. From these cone counts, the density of cones was calculated for a set of fixed distances from the fovea for locations throughout the image.
Cone photoreceptor packing density decreased from 27,712 cells/mm2 to 7,070 cells/mm2 from the retinal eccentricity of 0.30mm to 3.40mm along the superior meridian in five emmetropic eyes. Cone photoreceptor packing density in cells/mm2 was significantly lower in myopic eyes than in emmetropic eyes. At a given location, there was considerable individual variation in cone photoreceptor packing density, although more than 20% of the variance could be accounted for by differences in axial length.
Our results provide a baseline analysis of individual difference in cone photoreceptor packing density in healthy human eyes. As predicted by retinal stretching models, cone photoreceptor packing density is lower in highly myopic eyes than in emmetropic eyes.
Adaptive optics; cone photoreceptors; myopia; emmetropia