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James Feeks, Sarah Walters, Christina Schwarz, Jennifer J Hunter; Two-photon fluorescence lifetime ophthalmoscopy of intrinsic fluorophores on a cellular scale in the living macaque. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3431. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Fluorescence lifetime imaging quantifies the decay times of fluorophores and is sensitive to the environment of the fluorophore, which may be useful for early detection of changes in cell health. By combining fluorescence lifetime imaging with adaptive optics two-photon excited fluorescence ophthalmoscopy, we provide a novel technique to interrogate at a cellular level the properties of intrinsic fluorophores involved in cellular metabolism and the visual cycle.
For adaptive optics fluorescence lifetime imaging ophthalmoscopy (AOFLIO), a single photon counting detector and time-correlated single photon counting module were added to the fluorescence detection path of a two-photon adaptive optics scanning light ophthalmoscope. AOFLIO was performed in 2 macaques. Fluorescence was excited using 7 mW of 730 nm light and emission <550 nm collected for 150 s. Fluorescence lifetimes were determined for each pixel containing >500 photons by fitting exponential decay curves to photon arrival time histograms. Cones and rod regions were identified using the simultaneously collected fluorescence intensity image. For comparison, fluorescence lifetime images of fixed macaque retina were captured using the microscope arm of the same instrument. Wilcoxon rank-sum test was used to test for significance.
AOFLIO of the photoreceptor mosaic revealed significantly different (p = 0.001) mean lifetimes for cones (260 ± 60 ps; n = 731 cones) and rod regions (200 ± 18 ps; 8 locations in 2 macaques) (fig. 1). Some cones were difficult to distinguish in the intensity image but were identifiable in the lifetime image. In contrast, lifetime measurements of fixed macaque rods and cones showed no significant differences (p = 0.75).
AOFLIO using two-photon excitation makes possible measurements of the fluorescence lifetime of intrinsic retinal fluorophores at a cellular scale in the living macaque. AOFLIO revealed differences in fluorescence lifetime between rods and cones in vivo but not in fixed retina, emphasizing the importance of investigating retinal processes in their natural environment. AOFLIO may be a new method for distinguishing cell classes in the retina by their fluorophore composition or environment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Fig. 1. Two-photon excited fluorescence images of in vivo macaque photoreceptors. Cones can be distinguished by their longer fluorescence lifetime.
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