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Robin Sharma, Christina Schwarz, Grazyna Palczewska, Krzysztof Palczewski, David R Williams, Jennifer J Hunter; Quantification of retinol production and removal in photoreceptors in the living primate eye using two-photon ophthalmoscopy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2213. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Two-photon autofluorescence (TPAF) of all-trans-retinol in the outer retina can be used to quantify the kinetics of intermediate stages of the visual cycle. Our previous attempts at characterization of retinol kinetics in the living eye during dark adaptation were complicated by the effects of the two-photon imaging beam (Sharma et al., ARVO 2015). Here, we have bypassed the need for dark adaptation and developed a rapid approach for measuring the production and removal of retinol under steady illumination.
A two-photon adaptive optics scanning light ophthalmoscope was used for imaging photoreceptors in two macaques. TPAF was excited at 730 nm at 7 mW and emission was recorded between 400-550 nm. 35 s after the onset of the imaging beam, a 640 nm light flash of 1-4 s duration was delivered to produce an additional increase and subsequent decrease in TPAF. The time course of the TPAF response to the incremental flash was fit with exponential functions to extract rate constants.
The TPAF response of cones resolved in the image was weak because the cones were already bleached by the imaging beam. The rod response was much stronger, increasing rapidly with an exponential time constant of 1.2±0.5 s, and subsequently declining with a time constant of 76.6±7.2 s. This rate of retinol clearance measured under steady illumination is 1.6X faster than our previous measurements during dark adaptation in primates. The rate of retinol removal was faster than production, in agreement with previous studies in salamander, frog and mouse rods (Ala-Laurila et al., 2006, Wu et al., 2007, Chen et al., 2009). Despite the high light levels used for imaging, TPAF responses were repeatable over multiple trials and no detectable structural changes were observed in photoreceptor images.
This imaging scheme permits direct investigation of the pathways responsible for formation and removal of all-trans-retinol in rods. This provides a quantifiable measure of the visual cycle, without the need for complete dark adaptation, which is a shortcoming of conventional methods. This approach facilitates the use of in vivo two-photon ophthalmoscopy for rapid, objective assessment of photoreceptor function in the normal eye and in the diseased eye before and after pharmacological intervention.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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