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Khang T Huynh, Sarah Walters, Emma K. Foley, Keith Parkins, Jennifer J Hunter; Putative S cones in macaque identified by phasor analysis of adaptive optics fluorescence lifetime ophthalmoscopy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1778. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO) has revealed a difference in fluorescence lifetime between macaque cones and rods, likely due to functional differences. Similarly, S cones are morphologically and functionally disparate from M/L cones and may exhibit different fluorescence lifetimes. We apply phasor analysis to AOFLIO images to study the lifetime signatures of putative S cones, using their increased susceptibility to damage by multiphoton excitation (Schwarz et al. IOVS, in press) for identification.
Using 730 nm excitation, AOFLIO was performed on ≤1.1 x 1.3° regions in the photoreceptor layer of macaques (3 male, 1 female; 5-9 y.o.; 26 loc.; 4-20° ecc.; 2 min; 3.5 or 7 mW). Two locations were imaged twice at 7 mW: the first preferentially damaged S cones and the second was used only to identify them based on a fluorescence intensity decrease. Phasor analysis was performed by converting the decay trace at each pixel to a Cartesian coordinate (G,S) defined by its real and imaginary Fourier components. To identify two distinct cone populations, cones were masked at 4 locations and a Gaussian mixture model (GMM) analyzed the average (G,S) of each cone.
Across eccentricities and animals, phasor analysis of AOFLIO photoreceptor images produced a characteristic elliptical distribution of phasor coordinates (Fig. 1a-b) with distinct regions corresponding to cones and rods (Fig. 1c-d), confirming our previous work analyzing mean lifetimes. We also observed that cone pixels on the phasor plots appeared to separate into two elliptical distributions in 88% of locations (Fig. 1d). GMM classification of two cone populations resulted in one group corresponding to 11±0.66% of cones, consistent with S cone distribution. In two such locations, this population corresponded to cones subsequently identified as damaged by multiphoton excitation (87% sensitivity, 99% specificity).
By combining AOFLIO and phasor analysis, we identified a subset of cones consistent with S cone distribution and corroborated by their susceptibility to photodamage with multiphoton excitation. We do not know whether the different lifetime signature for putative S cones results from inherent morphological differences or a multiphoton absorption effect. By either mechanism, this approach represents a new way to functionally characterize cone photoreceptors.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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