Jennifer J. Hunter - US grants

Project Summary We wish to understand two important processes that sustain human vision; the visual cycle is responsible for regeneration of photopigment bleached by absorption of visible light and cellular metabolism is required in every living cell to provide energy. To study these processes, we need a method to visualize individual cells and measure molecular dynamics in the living eye. Some of the molecules involved are intrinsically fluorescent, but are inaccessible in the living eye with single- photon fluorescence imaging because the excitation falls outside the range of radiation that can penetrate the optics of the eye. By using considerably longer excitation wavelengths, two-photon excited fluorescence imaging has the potential to excite these otherwise inaccessible fluorophores and provide intrinsic contrast for imaging a number of retinal structures. In our initial experiments, we used an adaptive optics scanning light ophthalmoscope (AOSLO) to image two-photon fluorescence from cone inner segments in the living macaque eye (Hunter et al, 2011). By correcting the longitudinal chromatic aberration and material dispersion of the eye, we plan to improve the efficiency of our imaging systems and develop methodology for reliably imaging both structure and function of multiple retinal layers in the eye. Not only will this capability provide insight into normal cell mosaics and their biochemical processes, it will also improve our understanding of many diseases that impact these retinal biochemical cascades such as Stargardt's disease, macular degeneration and Leber's hereditary optic neuropathy.

PROJECT SUMMARY/ABSTRACT A highly collaborative team at the University of Rochester led by Jennifer Hunter and David Williams will partner with Grazyna Palczewska and Kris Palczewski at Polgenix, Inc. to translate in vivo two-photon excited fluorescence ophthalmoscopy (TPEFO) from monkey to human. This technology has the potential to provide new information on microscopic retinal morphology and to serve as a superior, objective measure to assess retinal function, a rare capability among existing imaging technologies. We will clarify if time-varying TPEF is tracking retinol by pharmacologically manipulating the visual cycle in monkey which might also serve as a model of human retinal disease. Effects of IR autofluorescence reduction, currently the only noticeable, consequence of TPEFO will be characterized. Additionally, we will optimize the light delivery of TPEFO intended to improve ocular safety but also establish thresholds for retinal damage. Monitoring retinal health in cortically blind human subjects after repeated TPEF imaging will establish whether TPEFO can be applied safely to normal human eyes.

IMAGING CORE SUMMARY The Imaging Core provides CVS faculty with access to equipment and highly trained staff that support histology, human and animal imaging, and adaptive optics for psychophysics and retinal imaging. The Imaging Core is sub-divided into three facilities. The Histology/Microscopy Facility supports brain and ocular tissue harvesting, processing, staining and microscopic examination (qualitative and quantitative). It is staffed by Tracy Bubel with over 24 years of experience in this area. The In Vivo Imaging Facility provides access to instruments for anterior segment, retinal, and brain imaging in addition to functional assessments of vision. The Adaptive Optics (AO) Facility consists of cutting-edge instrumentation utilising adaptive optics for visual psychophysics and cellular-scale retinal imaging. The AO Facility is staffed by two highly qualified engineers, Jie Zhang, Ph.D., and Qiang Yang, Ph.D. Through these three Facilities, the Imaging Core meets the expanding imaging requirements for advanced vision research and clinical research applications by CVS Core users.

Project Summary The retinal pigment epithelium (RPE) is critical to maintaining the health and normal function of photoreceptors, and is therefore involved in many retinal diseases that cause blindness. Throughout life, RPE cells accumulate waste products that cluster into fluorescent lipofuscin granules. In Stargardt disease, there is an acceleration of lipofuscin accumulation and changes in the molecular composition of the lipofuscin granules. The fluorescence of lipofuscin makes it possible to visualize the mosaic of RPE cells with an adaptive optics scanning light ophthalmoscope. However, characterizing RPE cells from structure and intensity alone does not provide sufficient information about the health of the cells. Measurement of the time delay in fluorescence emitted from RPE is related to the nature of the fluorophores and their environment including the composition of lipofuscin. Development of adaptive optics fluorescence lifetime ophthalmoscopy (AOFLIO) of the human RPE mosaic will provide an important tool to characterize RPE cells in both healthy and diseased eyes, where structural and functional biomarkers may be used for RPE evaluation, as well as diagnosing and monitoring disease. This proposal aims to develop and establish the ability of AOFLIO to detect cellular-level changes across the macula associated with healthy aging and Stargardt disease. Fluorescence lifetime will be measured across the retina by comparing AOFLIO in adult human subjects ranging in age from 20 to 70 years. The ability to identify RPE layer fluorescence lifetime changes in patients with Stargardt disease (10-30 years) and progression over 3 years will be assessed by longitudinal AOFLIO measurements in regions of atrophy, at the transition zone and in clinically-normal retina. Some of these subjects will also be imaged with a clinical prototype for widefield fluorescence lifetime imaging ophthalmoscopy (FLIO), generating one-of-a-kind comparative data. In addition, the current AOFLIO instrumentation will be replaced with innovative new technology designed to improve light efficiency and resolve finer features than currently possible. This project aims to evaluate the performance of AOFLIO, preparing us for future investigations to establish AOFLIO as an important measure of biomarkers of retinal degenerative disease, establishing AOFLIO as a prospective endpoint for use in clinical trials by providing rapid feedback on the effects of potential new therapies.