Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase.

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium.

Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4-/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4-/- mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.

Loss of the K+ channel Kv2.1 greatly reduces outward dark current and causes ionic dysregulation and degeneration in rod photoreceptors.

Vertebrate retinal photoreceptors signal light by suppressing a circulating "dark current" that maintains their relative depolarization in the dark. This dark current is composed of an inward current through CNG channels and NCKX transporters in the outer segment that is balanced by outward current exiting principally from the inner segment. It has been hypothesized that Kv2.1 channels carry a predominant fraction of the outward current in rods. We examined this hypothesis by comparing whole cell, suction electrode, and electroretinographic recordings from Kv2.1 knockout (Kv2.1-/-) and wild-type (WT) mouse rods. Single cell recordings revealed flash responses with unusual kinetics, and reduced dark currents that were quantitatively consistent with the measured depolarization of the membrane resting potential in the dark. A two-compartment (outer and inner segment) physiological model based on known ionic mechanisms revealed that the abnormal Kv2.1-/- rod photoresponses arise principally from the voltage dependencies of the known conductances and the NCKX exchanger, and a highly elevated fraction of inward current carried by Ca2+ through CNG channels due to the aberrant depolarization. Kv2.1-/- rods had shorter outer segments than WT and dysmorphic mitochondria in their inner segments. Optical coherence tomography of knockout animals demonstrated a slow photoreceptor degeneration over a period of 6 mo. Overall, these findings reveal that Kv2.1 channels carry 70-80% of the non-NKX outward dark current of the mouse rod, and that the depolarization caused by the loss of Kv2.1 results in elevated Ca2+ influx through CNG channels and elevated free intracellular Ca2+, leading to progressive degeneration.

In Situ Morphologic and Spectral Characterization of Retinal Pigment Epithelium Organelles in Mice Using Multicolor Confocal Fluorescence Imaging.

Purpose: To investigate the major organelles of the retinal pigment epithelium (RPE) in wild-type (WT, control) mice and their changes in pigmented Abca4 knockout (Abca4-/-) mice with in situ morphologic, spatial, and spectral characterization of live ex vivo flat-mounted RPE using multicolor confocal fluorescence microscopy (MCFM). Methods: In situ imaging of RPE flat-mounts of agouti Abca4-/- (129S4), agouti WT (129S1/SvlmJ) controls, and B6 albino mice (C57BL/6J-Tyrc-Brd) was performed with a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405 nm, 488 nm, 561 nm, and 640 nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software. Results: The 3-dimensional multicolor confocal images provided a detailed visualization of the RPE cell mosaic, including its melanosomes and lipofuscin granules, and their varying characteristics in the different mice strains. The autofluorescence spectra, spatial distribution, and morphologic features of melanosomes and lipofuscin granules were measured. Increased numbers of lipofuscin and reduced numbers of melanosomes were observed in the RPE of Abca4-/- mice relative to controls. Conclusions: A detailed assessment of the RPE autofluorescent granules and their changes ex vivo was possible with MCFM. For all excitation wavelengths, autofluorescence from the RPE cells was predominantly contributed by lipofuscin granules, while melanosomes were found to be essentially nonfluorescent. The red shift of the emission peak confirmed the presence of multiple chromophores within lipofuscin granules. The elevated autofluorescence levels in Abca4-/- mice correlated well with the increased number of lipofuscin granules.

Measurement of Diurnal Variation in Rod Outer Segment Length In Vivo in Mice With the OCT Optoretinogram.

Purpose: To investigate diurnal variation in the length of mouse rod outer segments in vivo. Methods: The lengths of rod inner and outer segments (RIS, ROS) of dark-adapted albino mice maintained on a 12-hour dark:12-hour light cycle with light onset 7 AM were measured at prescribed times (6:30 AM, 11 AM, 3:30 PM) during the diurnal cycle with optical coherence tomography (OCT), taking advantage of increased visibility, after a brief bleaching exposure, of the bands corresponding to RIS/ROS boundaries and ROS tips (ROST). Results: Deconvolution of OCT depth profiles resolved two backscatter bands located 7.4 ± 0.1 and 10.8 ± 0.2 µm (mean ± SEM) proximal to Bruch's membrane (BrM). These bands were identified with histology as arising from the apical surface of RPE and ROST, respectively. The average length of dark-adapted ROS at 6:30 AM was 17.7 ± 0.8 µm. By 11 AM, the average ROS length had decreased by 10% to 15.9 ± 0.7 µm. After 11 AM, the ROS length increased steadily at an average rate of 0.12 µm/h, returning to baseline length by 23.5 hours in the cycle. Conclusions: The diurnal variation in ROS length measured in these experiments is consistent with prior histological investigations showing that rodent rod discs are phagocytosed by the RPE maximally over several hours around the time of normal light onset. The rate of recovery of ROS to baseline length before normal light onset is consistent with the hypothesis that disc membrane synthesis is fairly constant over the diurnal cycle.

Temporal speckle-averaging of optical coherence tomography volumes for in-vivo cellular resolution neuronal and vascular retinal imaging.

It has been recently demonstrated that structures corresponding to the cell bodies of highly transparent cells in the retinal ganglion cell layer could be visualized noninvasively in the living human eye by optical coherence tomography (OCT) via temporal averaging. Inspired by this development, we explored the application of volumetric temporal averaging in mice, which are important models for studying human retinal diseases and therapeutic interventions. A general framework of temporal speckle-averaging (TSA) of OCT and optical coherence tomography angiography (OCTA) is presented and applied to mouse retinal volumetric data. Based on the image analysis, the eyes of mice under anesthesia exhibit only minor motions, corresponding to lateral displacements of a few micrometers and rotations of a fraction of 1 deg. Moreover, due to reduced eye movements under anesthesia, there is a negligible amount of motion artifacts within the volumes that need to be corrected to achieve volume coregistration. In addition, the relatively good optical quality of the mouse ocular media allows for cellular-resolution imaging without adaptive optics (AO), greatly simplifying the experimental system, making the proposed framework feasible for large studies. The TSA OCT and TSA OCTA results provide rich information about new structures previously not visualized in living mice with non-AO-OCT. The mechanism of TSA relies on improving signal-to-noise ratio as well as efficient suppression of speckle contrast due to temporal decorrelation of the speckle patterns, enabling full utilization of the high volumetric resolution offered by OCT and OCTA.

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

Directional optical coherence tomography reveals melanin concentration-dependent scattering properties of retinal pigment epithelium.

Optical coherence tomography (OCT) is a powerful tool in ophthalmology that provides in vivo morphology of the retinal layers and their light scattering properties. The directional (angular) reflectivity of the retinal layers was investigated with focus on the scattering from retinal pigment epithelium (RPE). The directional scattering of the RPE was studied in three mice strains with three distinct melanin concentrations: albino (BALB/c), agouti (129S1/SvlmJ), and strongly pigmented (C57BL/6J). The backscattering signal strength was measured with a directional OCT system in which the pupil entry position of the narrow OCT beam can be varied across the dilated pupil of the eyes of the mice. The directional reflectivity of other retinal melanin-free layers, including the internal and external limiting membranes, and Bruch's membrane (albinos) were also measured and compared between the strains. The intensity of light backscattered from these layers was found highly sensitive to the angle of illumination, whereas the inner/outer segment (IS/OS) junctions showed a reduced sensitivity. The reflections from the RPE are largely insensitive in highly pigmented mice. The differences in directional scattering between strains shows that directionality decreases with an increase in melanin concentrations in RPE, suggesting increasing contribution of Mie scattering by melanosomes.

Novel window for cancer nanotheranostics: non-invasive ocular assessments of tumor growth and nanotherapeutic treatment efficacy in vivo.

In cancer research there is a fundamental need for animal models that allow the in vivo longitudinal visualization and quantification of tumor development, nanotherapeutic delivery, the tumor microenvironment including blood vessels, macrophages, fibroblasts, immune cells, and extracellular matrix, and the tissue response to treatment. To address this need, we developed a novel mouse ocular xenograft model. Green fluorescent protein (GFP) expressing human glioblastoma cells (between 500 and 10,000) were implanted into the subretinal space of immunodeficient mice (56 eyes). The resultant xenografts were imaged in vivo non-invasively with combined fluorescence scanning laser ophthalmoscopy (SLO) and volumetric optical coherence tomography (OCT) for a period up to several months. Most xenografts exhibited a latent phase followed by a stable or rapidly increasing volume, but about 1/3 underwent spontaneous remission. After prescribed growth, a population of tumors was treated with intravenously delivered doxorubicin-containing porphyrin and cholic acid-based nanoparticles ("nanodox"). Fluorescence resonance energy transfer (FRET) emission (doxorubicin → porphyrin) was used to localize nanodox in the xenografts, and 690 nm light exposure to activate it. Such photo-nanotherapy was highly effective in reducing tumor volume. Histopathology and flow cytometry revealed CD4 + and CD8 + immune cell infiltration of xenografts. Overall, the ocular model shows potential for examining the relationships between neoplastic growth, neovascularization and other features of the immune microenvironment, and for evaluating treatment response longitudinally in vivo.

Aperture phase modulation with adaptive optics: a novel approach for speckle reduction and structure extraction in optical coherence tomography.

Speckle is an inevitable consequence of the use of coherent light in imaging and acts as noise that corrupts image formation in most applications. Optical coherence tomographic imaging, as a technique employing coherence time gating, suffers from speckle. We present here a novel method of suppressing speckle noise intrinsically compatible with adaptive optics (AO) for confocal coherent imaging: modulation of the phase in the system pupil aperture with a segmented deformable mirror (DM) to introduce minor perturbations in the point spread function. This approach creates uncorrelated speckle patterns in a series of images, enabling averaging to suppress speckle noise while maintaining structural detail. A method is presented that efficiently determines the optimal range of modulation of DM segments relative to their AO-optimized position so that speckle noise is reduced while image resolution and signal strength are preserved. The method is active and independent of sample properties. Its effectiveness and efficiency are quantified and demonstrated by both ex vivo non-biological and in vivo biological applications.

Effect of a contact lens on mouse retinal in vivo imaging: Effective focal length changes and monochromatic aberrations.

For in vivo mouse retinal imaging, especially with Adaptive Optics instruments, application of a contact lens is desirable, as it allows maintenance of cornea hydration and helps to prevent cataract formation during lengthy imaging sessions. However, since the refractive elements of the eye (cornea and lens) serve as the objective for most in vivo retinal imaging systems, the use of a contact lens, even with 0 Dpt. refractive power, can alter the system's optical properties. In this investigation we examined the effective focal length change and the aberrations that arise from use of a contact lens. First, focal length changes were simulated with a Zemax mouse eye model. Then ocular aberrations with and without a 0 Dpt. contact lens were measured with a Shack-Hartmann wavefront sensor (SHWS) in a customized AO-SLO system. Total RMS wavefront errors were measured for two groups of mice (14-month, and 2.5-month-old), decomposed into 66 Zernike aberration terms, and compared. These data revealed that vertical coma and spherical aberrations were increased with use of a contact lens in our system. Based on the ocular wavefront data we evaluated the effect of the contact lens on the imaging system performance as a function of the pupil size. Both RMS error and Strehl ratios were quantified for the two groups of mice, with and without contact lenses, and for different input beam sizes. These results provide information for determining optimum pupil size for retinal imaging without adaptive optics, and raise critical issues for design of mouse optical imaging systems that incorporate contact lenses.

Loss of cone function without degeneration in a novel Gnat2 knock-out mouse.

Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod-1 s-1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.

The discovery of the ability of rod photoreceptors to signal single photons.

Vertebrate rod photoreceptors evolved the astonishing ability to respond reliably to single photons. In parallel, the proximate neurons of the visual system evolved the ability to reliably encode information from a few single-photon responses (SPRs) as arising from the presence of an object of interest in the visual environment. These amazing capabilities were first inferred from measurements of human visual threshold by Hecht et al. (1942), whose paper has since been cited over 1,000 times. Subsequent research, in part inspired by Hecht et al.'s discovery, has directly measured rod SPRs, characterized the molecular mechanism responsible for their generation, and uncovered much about the specializations in the retina that enable the reliable transmission of SPRs in the teeth of intrinsic neuronal noise.

Photoreceptor Layer Thickness Changes During Dark Adaptation Observed With Ultrahigh-Resolution Optical Coherence Tomography.

Purpose: To examine outer retinal band changes after flash stimulus and subsequent dark adaptation with ultrahigh-resolution optical coherence tomography (UHR-OCT). Methods: Five dark-adapted left eyes of five normal subjects were imaged with 3-µm axial-resolution UHR-OCT during 30 minutes of dark adaptation following 96%, 54%, 23%, and 0% full-field and 54% half-field rhodopsin bleach. We identified the ellipsoid zone inner segment/outer segment (EZ[IS/OS]), cone interdigitation zone (CIZ), rod interdigitation zone (RIZ), retinal pigment epithelium (RPE), and Bruch's membrane (BM) axial positions and generated two-dimensional thickness maps of the EZ(IS/OS) to the four bands. The average thickness over an area of the thickness map was compared against that of the dark-adapted baselines. The time-dependent thickness changes (photoresponses) were statistically compared against 0% bleach. Dark adaptometry was performed with the same bleaching protocol. Results: The EZ(IS/OS)-CIZ photoresponse was significantly different at 96% (P < 0.0001) and 54% (P = 0.006) bleach. At all three bleaching levels, the EZ(IS/OS)-RIZ, -RPE, and -BM responses were significantly different (P < 0.0001). The EZ(IS/OS)-CIZ and EZ(IS/OS)-RIZ time courses were similar to the recovery of rod- and cone-mediated sensitivity, respectively, measured with dark adaptometry. The maximal EZ(IS/OS)-CIZ and EZ(IS/OS)-RIZ response magnitudes doubled from 54% to 96% bleach. Both EZ(IS/OS)-RPE and EZ(IS/OS)-BM responses resembled dampened oscillations that were graded in amplitude and duration with bleaching intensity. Half-field photoresponses were localized to the stimulated retina. Conclusions: With noninvasive, near-infrared UHR-OCT, we characterized three distinct, spatially localized photoresponses in the outer retinal bands. These photoresponses have potential value as physical correlates of photoreceptor function.

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130.

BACKGROUND: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice. METHODS: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor ß (gp-130) was injected intravitreally immediately after RD. RESULTS: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade. CONCLUSIONS: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

Bright flash response recovery of mammalian rods in vivo is rate limited by RGS9.

The temporal resolution of scotopic vision is thought to be constrained by the signaling kinetics of retinal rods, which use a highly amplified G-protein cascade to transduce absorbed photons into changes in membrane potential. Much is known about the biochemical mechanisms that determine the kinetics of rod responses ex vivo, but the rate-limiting mechanisms in vivo are unknown. Using paired flash electroretinograms with improved signal-to-noise, we have recorded the amplitude and kinetics of rod responses to a wide range of flash strengths from living mice. Bright rod responses in vivo recovered nearly twice as fast as all previous recordings, although the kinetic consequences of genetic perturbations previously studied ex vivo were qualitatively similar. In vivo, the dominant time constant of recovery from bright flashes was dramatically reduced by overexpression of the RGS9 complex, revealing G-protein deactivation to be rate limiting for recovery. However, unlike previous ex vivo recordings, dim flash responses in vivo were relatively unaffected by RGS9 overexpression, suggesting that other mechanisms, such as calcium feedback dynamics that are strongly regulated by the restricted subretinal microenvironment, act to determine rod dim flash kinetics. To assess the consequences for scotopic vision, we used a nocturnal wheel-running assay to measure the ability of wild-type and RGS9-overexpressing mice to detect dim flickering stimuli and found no improvement when rod recovery was speeded by RGS9 overexpression. These results are important for understanding retinal circuitry, in particular as modeled in the large literature that addresses the relationship between the kinetics and sensitivity of retinal responses and visual perception.

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s-1 Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10-5 cm⋅s-1, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

The Photosensitivity of Rhodopsin Bleaching and Light-Induced Increases of Fundus Reflectance in Mice Measured In Vivo With Scanning Laser Ophthalmoscopy.

PURPOSE: To quantify bleaching-induced changes in fundus reflectance in the mouse retina. METHODS: Light reflected from the fundus of albino (Balb/c) and pigmented (C57Bl/6J) mice was measured with a multichannel scanning laser ophthalmoscopy optical coherence tomography (SLO-OCT) optical system. Serial scanning of small retinal regions was used for bleaching rhodopsin and measuring reflectance changes. RESULTS: Serial scanning generated a saturating reflectance increase centered at 501 nm with a photosensitivity of 1.4 × 10-8 per molecule µm2 in both strains, 2-fold higher than expected were irradiance at the rod outer segment base equal to that at the retinal surface. The action spectrum of the reflectance increase corresponds to the absorption spectrum of mouse rhodopsin in situ. Spectra obtained before and after bleaching were fitted with a model of fundus reflectance, quantifying contributions from loss of rhodopsin absorption with bleaching, absorption by oxygenated hemoglobin (HbO2) in the choroid (Balb/c), and absorption by melanin (C57Bl/6J). Both mouse strains exhibited light-induced broadband reflectance changes explained as bleaching-induced reflectivity increases at photoreceptor inner segment/outer segment (IS/OS) junctions and OS tips. CONCLUSIONS: The elevated photosensitivity of rhodopsin bleaching in vivo is explained by waveguide condensing of light in propagation from rod inner segment (RIS) to rod outer segment (ROS). The similar photosensitivity of rhodopsin in the two strains reveals that little light backscattered from the sclera can enter the ROS. The bleaching-induced increases in reflectance at the IS/OS junctions and OS tips resemble results previously reported in human cones, but are ascribed to rods due to their 30/1 predominance over cones in mice and to the relatively minor amount of cone M-opsin in the regions scanned.

New Developments in Murine Imaging for Assessing Photoreceptor Degeneration In Vivo.

Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs' membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al., Vis Res 102:71-79, 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.