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Robin Sharma, Christina Schwarz, Grazyna Palczewska, Krzysztof Palczewski, David R Williams, Jennifer J Hunter; In vivo two-photon fluorescence kinetics of primate rods and cones during light and dark adaptation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5968.
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© ARVO (1962-2015); The Authors (2016-present)
There is substantial evidence from multiple species that the two-photon fluorescence (TPF) of photoreceptors increases following photopigment bleaching and declines during regeneration. We have investigated this phenomenon in living monkeys with the goals of characterizing the kinetics of this response independently in rods and cones and clarifying its molecular origin.
Three healthy macaques were imaged with a two-photon adaptive optics scanning light ophthalmoscope (λex 730 nm, λem 400-550 nm). Small patches of cells were bleached with 565 nm light (14 mW/cm2 for 5 s). TPF was then recorded after waiting for a variable period in the dark ranging from 0-30 min, corresponding to 0-100% initially available photopigment (IAP). To generate dark adaptation curves, initial TPF values were plotted as a function of time in the dark after bleach. To investigate light adaptation to the TPF excitation beam, TPF was tracked over 2 min for different IAP states.
During dark adaptation, rod TPF decreased exponentially with time constants ranging from 1.5-4 min. Cone TPF during dark adaptation showed an overall decrease, but was non-monotonic. Rod and cone TPF kinetics during light adaptation were different. Rod TPF trends strongly depended on initially available photopigment (IAP). For 0-15 % IAP (0-2 min after bleach), rod TPF decreased monotonically, and eventually plateaued. For 40-100 % IAP (>5 min after bleach), rod TPF always increased to a plateau. Cone TPF trends during light adaptation were consistent across all IAP conditions. Cone TPF increased to a peak within 5-15 s and then declined to a plateau. Approximately 5-8 % of the cones forming a fairly regular, sparse mosaic showed little or no increase in TPF following the bleach.
The sparse array of cones little affected by the bleach is consistent with the S cone mosaic in density and distribution. TPF kinetics differed from existing pigment regeneration models but were qualitatively consistent with the expected production and removal of retinoids later in the visual cycle. While TPF from other molecules cannot be excluded, these measurements are probably dominated by the kinetics of retinoids during light and dark adaptation, providing a way to quantify stages of the retinoid cycle that have been otherwise inaccessible in the living primate eye.
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