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CLEVELAND, OH, United States

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 188.13K | Year: 2010

DESCRIPTION (provided by applicant): Our long-term goal is to develop a clinical instrument, the 2-photon ophthalmoscope, for non-invasive, high-resolution and repetitive imaging of biochemical processes within the human retina. Such an instrument will have tremendous potential for early detection of age and disease related changes in the eye, long before pathological manifestations of retinal disease become obvious. This real-time retinal imaging instrument will also be critical for rapid evaluation of various pharmacological agents used to treat retinal pathologies. The method has the great advantage of imaging endogenous retinoid fluorophores in their native state without the need for additional staining. In Phase I, we seek to answer whether two-photon excitation based imaging can track age related changes in the retina and then whether a 2-photon ophthalmoscope, with the ultimate goal of clinical instrument can be made. We are proposing three specific aims: (1) Determine whether two-photon excitation imaging can be used to monitor age related changes in human retina; (2) Image the eye of a living monkey to determine the feasibility of two-photon adaptive optics ophthalmoscope system for non-invasive, in vivo imaging of human retinal pigment epithelium cells; and (3) Determine if the size and cost of the instrument could be reduced by replacing the Ti:Sapphire modelocked laser with a femtosecond fiber laser to validate commercialization plans. Once these aims are fulfilled, in Phase 2 we will use the derived data to adapt the two-photon adaptive optics ophthalmoscope for imaging human eyes in vivo and characterize two-photon fluorescence in eyes affected by retina diseases. In addition, the use of the fiber laser and micro-electro- mechanical systems deformable mirror technology, in adaptive optics design, promises to reduce the cost and allow the physical footprint of the instrument to be kept small, greatly aiding potential commercialization. PUBLIC HEALTH RELEVANCE: We seek to develop a novel instrument for noninvasive imaging of the back of the eye with sub-cellular resolution. The instrument will visualize the age or disease related changes in the biochemical processes within human retina, specifically retinoid cycle. Our goal is to further understanding of the biochemistry of vision to allow for rapid evaluation of various pharmacological interventions to prevent retinal degeneration and other pathologies at the early stages, before the retina degenerates and vision is irreparably damaged.

Palczewska G.,Polgenix, Inc. | Maeda T.,Case Western Reserve University | Imanishi Y.,Case Western Reserve University | Sun W.,Polgenix, Inc. | And 6 more authors.
Nature Medicine

Multiphoton excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in subcellular structures called retinosomes mediate regeneration of the visual chromophore, 11-cis-retinal, by the visual cycle. But harmful fluorescent condensation products of retinoids also occur in the retina. We report that in wild-type mice, excitation with a wavelength of ∼730 nm identified retinosomes in the retinal pigment epithelium, and excitation with a wavelength of ∼910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older wild-type mice and mice with defective clearance of all-trans-retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age-related macular degeneration and other defects in retinoid metabolism. © 2010 Nature America, Inc. All rights reserved. Source

Sharma R.,University of Rochester | Schwarz C.,University of Rochester | Williams D.R.,University of Rochester | Palczewska G.,Polgenix, Inc. | And 2 more authors.
Investigative Ophthalmology and Visual Science

PURPOSE. The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Twophoton fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. METHODS. We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye’s pupil. RESULTS. The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. CONCLUSIONS. Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas. © 2016, Association for Research in Vision and Ophthalmology Inc. All rights reserved. Source

Palczewska G.,Polgenix, Inc. | Vinberg F.,University of Washington | Stremplewski P.,Nicolaus Copernicus University | Bircher M.P.,University of Bern | And 7 more authors.
Proceedings of the National Academy of Sciences of the United States of America

Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400- to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experimentswith rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by twophoton isomerization of visual pigments. Source

Orban T.,Case Western Reserve University | Palczewska G.,Polgenix, Inc. | Palczewski K.,Case Western Reserve University
Journal of Biological Chemistry

Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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