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.

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.

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.

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).

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.

Temporal speckle-averaging of optical coherence tomography volumes for 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.

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.

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 mechanism of photon-like dark noise in rod photoreceptors.

Pugh highlights recent work ruling out a role for ultraweak photon emission in spontaneous photon-like events in retinal rods.

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.

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

In cancer research there is a fundamental need for animal models that allow the 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).

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.

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.

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.

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.

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.

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 (Gα), revealing these responses to be triggered by phototransduction.

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.

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.

In vivo wide-field multispectral scanning laser ophthalmoscopy-optical coherence tomography mouse retinal imager: longitudinal imaging of ganglion cells, microglia, and Müller glia, and mapping of the mouse retinal and choroidal vasculature.

Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) provide complementary views of the retina, with the former collecting fluorescence data with good lateral but relatively low-axial resolution, and the latter collecting label-free backscattering data with comparable lateral but much higher axial resolution. To take maximal advantage of the information of both modalities in mouse retinal imaging, we have constructed a compact, four-channel, wide-field (∼50  deg) system that simultaneously acquires and automatically coregisters three channels of confocal SLO and Fourier domain OCT data. The scanner control system allows “zoomed” imaging of a region of interest identified in a wide-field image, providing efficient digital sampling and localization of cellular resolution features in longitudinal imaging of individual mice.

Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO.

Scanning laser ophthalmoscopy (SLO) employs the eye's optics as a microscope objective for retinal imaging in vivo. The mouse retina has become an increasingly important object for investigation of ocular disease and physiology with optogenetic probes. SLO imaging of the mouse eye, in principle, can achieve submicron lateral resolution thanks to a numerical aperture (NA) of ∼0.

Photoreceptor disc morphogenesis: The classical evagination model prevails.

Vision begins in photoreceptor outer segments with light captured by opsins in continually synthesized disc membranes. The process by which rod photoreceptor discs are formed has been controversial. In this issue, Ding et al.

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.

Wavefront correction and high-resolution in vivo OCT imaging with an objective integrated multi-actuator adaptive lens.

Adaptive optics is rapidly transforming microscopy and high-resolution ophthalmic imaging. The adaptive elements commonly used to control optical wavefronts are liquid crystal spatial light modulators and deformable mirrors. We introduce a novel Multi-actuator Adaptive Lens that can correct aberrations to high order, and which has the potential to increase the spread of adaptive optics to many new applications by simplifying its integration with existing systems.

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess.