Edward N Pugh, Jr.

• Doctor of Philosophy
• Professor at University of California, Davis

Purpose: To investigate differences in fundus autofluorescence (AF) spectra of pigmented (C57Bl/6) and albino (Balb/c) mouse retinas. Methods: AF spectra were measured with a scanning laser ophthalmoscope (SLO) with a high-resolution spectrometer. The action spectrum of a 635 nm AF "spike" in albino mice was measured to estimate the underlying a...

To better understand the molecular basis of the mouse electroretinogram (ERG) we have developed a biophysical model of the rod photoreceptor layer s ionic mechanisms and applied current source-density (CSD) analysis to predict the genetically and pharmacologically isolated rod ERG a-wave. The saturating a-wave is characterized by a rapid relaxation...

A goal of contemporary physiology is to translate the knowledge obtained ex vivo of the molecular structure and function of ionic mechanisms into tools for quantitative, in vivo measurement of that function in humans, and in animal models of disease and therapeutic intervention. Non-invasive field potentials such as ECGs, EMGs, EEGs, and ERGs hold...

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...

Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of granular pigmented organelles found in the retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin granules in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degener...

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...

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 m...

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 o...

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 ave...

Significance Microglia, the resident macrophages of the central nervous system, are critical for synaptic pruning and maintenance and for mitigating injury and neurodegeneration. Determining whether microglia–neuron interactions are beneficial in specific instances has been difficult, largely because of the local and transient nature of the interac...

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 w...

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

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 adap...

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 t...

Speckle is an inevitable consequence of the use of coherent light in optical coherence tomography (OCT), and often acts as noise that obscures micro-structures of biological tissue. We here present a novel method of suppressing speckle noise intrinsically compatible with adaptive optics (AO) in OCT system: by modulating the phase inside the imaging...

To present our in vivo observations on angular dependence of mouse retina layers reflectivity measured by directional mouse retinal OCT system. To quantify and model differences observed between angular dependent reflectivity in inner and outer retina layers in different mouse strains.

To evaluate feasibility of in vivo retinal imaging methods for monitoring of Photoreceptor-RPE-Choroid Neurovascular Unit (PRC-NVU) morphology and function locally and longitudinally in individual animals during aging.

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 ca...

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...

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 capab...

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-fiel...

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 investigat...

Significance Complete activation of the phototransduction G-protein cascade of dark-adapted rod photoreceptors causes outer segments to undergo 10% elongation and large local increases in backscattering, as measured in vivo with noninvasive, high-resolution optical coherence tomography. Maximal elongation is caused by a potentially harmful 20% incr...

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...

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 quantitativel...

We have successfully used the mouse eye as a non-surgical window for highly efficient, optical investigation of xenograft models, using a state-of-the-art ocular imaging facility, the UC Davis “EyePod”. The EyePod employs single-cell resolution intravital confocal microscopy and optical coherence tomography, performed completely non-invasively thro...

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...

Chorioretinal blood vessel morphology in mice is of great interest to researchers studying eye disease mechanisms in animal models. Two leading retinal imaging modalities -- Optical Coherence Tomography (OCT) and Scanning Laser Ophthalmoscopy (SLO) -- have offered much insight into vascular morphology and blood flow. OCT “flow-contrast” methods hav...

We present a novel system for adaptive optics two photon imaging. We utilize the bandwidth of the femtosecond excitation beam to perform coherence gated imaging (OCT) of the sample. The location of the focus is directly observable in the cross sectional OCT images, and adjusted to the desired depth plane. Next, using real time volumetric OCT, we pe...

We present a new type of adaptive lens with 18 actuators that can correct up the 4th order of aberration. The Multi-actuator Adaptive Lens (M-AL) can guarantee a good level of aberration correction for many applications and, with respect to deformable mirror, it allows the realization of more compact and simple optical systems. The adaptive lens is...

Scanning Laser Ophthalmoscopy (SLO) is a very important imaging tool in ophthalmology research. By combing with Adaptive Optics (AO) technique, AO-SLO can correct for ocular aberrations resulting in cellular level resolution, allowing longitudinal studies of single cells morphology in the living eyes. The numerical aperture (NA) sets the optical re...

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 a...

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...

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. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201508093) show conclusively that rod discs are formed by plasma membrane...

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...

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 cellul...

A compact, non-invasive multi-modal system has been developed for in vivo mouse retina imaging. It is configured for simultaneously detecting green and red fluorescent protein signals with scanning laser ophthalmoscopy (SLO) back-scattered light from the SLO illumination beam, and depth information about different retinal layers by means of Optical...

We present a novel adaptive lens that can correct high order aberrations, and which has the potential to increase the diffusion of adaptive optics to many new applications by simplifying the integration of a wavefront corrector inside existing systems. The adaptive lens design that we present can correct for Zernike wavefront aberrations up to the...

Vertebrate vision begins when retinal photoreceptors transduce photons into electrical signals that are then relayed to other neurons in the eye, and ultimately to the brain. In rod photoreceptors, transduction of single photons is achieved by a well-understood G-protein cascade that modulates cGMP levels, and in turn, cGMP-sensitive inward current...

Mutations in RPE65 or lecithin-retinol acyltransferase (LRAT) disrupt 11-cis-retinal synthesis and cause Leber congenital amaurosis (LCA), a severe hereditary blindness occurring in early childhood. The pathology is attributed to a combination of 11-cis-retinal deficiency and photoreceptor degeneration. The mistrafficking of cone membrane-associate...

Key points The early receptor potential (ERP) of the mouse electroretinogram (ERG) was measured in wild‐type (WT) mice, in mice ( Opn1sw −/− ) that lack S‐cone opsin and overexpress M‐cone opsin, and in mice heterozygous for the retinal pigment epithelium isomerase Rpe65. The amplitude of the ERP saturated exponentially with flash intensity. In WT...

Optical coherence tomography is an imaging modality that is broadly used in ophthalmic diagnostics. The current generation of OCT systems enables reliable acquisition of volumetric scans containing information about the thicknesses of the various retinal layers. Thus, monitoring layer thickness changes is the main quantitative analysis performed by...

We demonstrate Adaptive optics - Optical Coherence Tomography (OCT) with modal sensorless Adaptive Optics correction with the use of novel Adaptive Lens (AL) applied for in-vivo imaging of mouse retinas. The AL can generate low order aberrations: defocus, astigmatism, coma and spherical aberration that were used in an adaptive search algorithm. Acc...

Animal models of human diseases play an important role in studying and advancing our understanding of these conditions, allowing molecular level studies of pathogenesis as well as testing of new therapies. Recently several non-invasive imaging modalities including Fundus Camera, Scanning Laser Ophthalmoscopy (SLO) and Optical Coherence Tomography (...

The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial vari...

Shadow is an inseparable aspect of all natural scenes. When there are multiple light sources or multiple reflections several different shadows may overlap at the same location and create complicated patterns. Shadows are a potentially good source of information about a scene if the shadow regions can be properly identified and segmented. However, s...

Purpose: We measured the bleaching and regeneration kinetics of rhodopsin in the living human eye with two-wavelength, wide-field scanning laser ophthalmoscopy (SLO), and investigated the effect of rhodopsin bleaching on autofluorescence intensity. Methods: The retina was imaged with an Optos P200C SLO by its reflectance of 532 and 633 nm light,...

Rod photoreceptors generate amplified, reproducible responses to single photons via a G protein signaling cascade. Surprisingly, genetic perturbations that dramatically alter the deactivation of the principal signal amplifier, the GPCR rhodopsin (R(∗)), do not much alter the amplitude of single-photon responses (SPRs). These same perturbations, whe...

In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shift...

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and t...

Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57Bl/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-o...

### Associate Editor: Kenton J. Swartz The JGP is delighted to announce that Kenton Swartz has joined the group of associate editors. Kenton is a senior investigator in the Molecular Physiology and Biophysics Section of the Basic Neuroscience Program at the National Institute of Neurological

This past summer I was contacted by a colleague who had just attended the [Gordon Conference on Ion Channels][1]. The glowing account given of the conference ranked it as exceptional: outstanding speakers, exciting research, and great scientific interactions. Other attendees with whom I spoke rated

Visual thresholds of mice for the detection of small, brief targets were measured with a novel behavioral methodology in the dark and in the presence of adapting lights spanning ∼8 log(10) units of intensity. To help dissect the contributions of rod and cone pathways, both wild-type mice and mice lacking rod (Gnat1(-/-)) or cone (Gnat2(cpfl3)) func...

### Introduction The purpose of the Perspectives in General Physiology is to provide a forum where scientific uncertainties or controversies are discussed in an authoritative, yet open, manner. Perspectives are solicited by the editors—often based on recommendations by members of the editorial

Phototransduction in retinal rods is one of the most extensively studied G-protein signaling systems. In recent years, our understanding of the biochemical steps that regulate the deactivation of the rod's response to light has greatly improved. Here, we summarize recent advances and highlight some of the remaining puzzles in this model signaling s...

Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme....