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 ito 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.

Public Health Relevance

My new laboratory at the University of Rochester will develop two-photon fluorescence imaging into a highly efficient technique to visualize non-invasively for the first time neural cells in the back of the eye and to monitor important cellular processes in the living eye. This research could lead to improvements in the diagnosis of some retinal diseases, monitoring of their progression and establishing the efficacy of treatment strategies.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-NT-L (09))
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Neuhold, Lisa
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University of Rochester
Schools of Dentistry
United States
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Schwarz, Christina; Sharma, Robin; Cheong, Soon Keen et al. (2018) Selective S Cone Damage and Retinal Remodeling Following Intense Ultrashort Pulse Laser Exposures in the Near-Infrared. Invest Ophthalmol Vis Sci 59:5973-5984
Sharma, Robin; Schwarz, Christina; Hunter, Jennifer J et al. (2017) Formation and Clearance of All-Trans-Retinol in Rods Investigated in the Living Primate Eye With Two-Photon Ophthalmoscopy. Invest Ophthalmol Vis Sci 58:604-613
Marcos, Susana; Werner, John S; Burns, Stephen A et al. (2017) Vision science and adaptive optics, the state of the field. Vision Res 132:3-33
Rossi, Ethan A; Granger, Charles E; Sharma, Robin et al. (2017) Imaging individual neurons in the retinal ganglion cell layer of the living eye. Proc Natl Acad Sci U S A 114:586-591
Schwarz, Christina; Sharma, Robin; Fischer, William S et al. (2016) Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans. Biomed Opt Express 7:5148-5169
Sharma, Robin; Williams, David R; Palczewska, Grazyna et al. (2016) Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye. Invest Ophthalmol Vis Sci 57:632-46
Sharma, Robin; Schwarz, Christina; Williams, David R et al. (2016) In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones. Invest Ophthalmol Vis Sci 57:647-57
Masella, Benjamin D; Hunter, Jennifer J; Williams, David R (2014) New wrinkles in retinal densitometry. Invest Ophthalmol Vis Sci 55:7525-34
Palczewska, Grazyna; Golczak, Marcin; Williams, David R et al. (2014) Endogenous fluorophores enable two-photon imaging of the primate eye. Invest Ophthalmol Vis Sci 55:4438-47
Masella, Benjamin D; Hunter, Jennifer J; Williams, David R (2014) Rod photopigment kinetics after photodisruption of the retinal pigment epithelium. Invest Ophthalmol Vis Sci 55:7535-44

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