In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones

Abstract

PurposeThe retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates.MethodsWe developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye's pupil.ResultsThe fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution.ConclusionsAlthough other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas.

Imaging individual neurons in the retinal ganglion cell layer of the living eye

“…Experiments were carried out on three different AOSLO systems capable of simultaneous confocal and off-axis detection: a human research AOSLO (48,49), a compact commercial prototype human AOSLO (50), and a nonhuman primate two-photon AOSLO (TPAOSLO) (26,51). Some humans were imaged on both systems, permitting comparison between systems, but some major differences between them prevented direct comparisons (see SI Materials and Methods for details).…”

Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans

“…After this adaptation period, the retinal location was exposed for either 40 s to the minimally required power of 0.5 mW (20.4 J/cm 2 ) or 80 s to 1 mW (81.7 J/cm 2 ) of the pulsed laser light. In previous work [26], at a similar laser power of 0.75 mW, TPEF was observed to increase after dark adapation to a plateau due to stimulation of the imaging laser itself. A minimum duration of 40 s was chosen to allow the TPEF to reach the plateau.…”

“…The instrument has been described in detail elsewhere [25,26]. The TP-AOSLO employed three light sources in the near IR: a laser diode (QFLD-850-20S, QPhotonics, Ann Arbor, MI, USA) emitting at 840 nm with maximum power output at the system's exit pupil of 50 µW for wavefront sensing, a superluminescent diode (SLD; S-790-G-I-15, Superlum, Carrigtohill, Ireland) emitting at 790 nm with maximum power output of 250 µW for confocal reflectance imaging and a pulsed Ti:Sapphire laser (MaiTai XF-1 with DeepSee attachment for dispersion compensation, Spectra-Physics, Santa Clara, CA, USA) with a tunable central wavelength between 710 nm and 920 nm for two-photon excitation and confocal reflectance imaging.…”

“…Multiphoton excitation [14][15][16] offers a special advantage for ophthalmoscopy, because important fluorophores such as NAD(P)H [17][18][19] and retinoids [20,21] can only be accessed with UV light, a spectral range that is blocked by the anterior optics of the human eye [22] and raises concerns over light safety [23]. Recently, adaptive optics two-photon excitation fluorescence (TPEF) ophthalmoscopy has been demonstrated in the living macaque eye [24][25][26]. Many cell types throughout the retina, including ganglion cells, could be visualized, with photoreceptor outer segments providing the strongest signal [25].…”

“…Deficiency in any of these steps due to genetic mutations, dietary factors or age will result in slowed photopigment regeneration [28], retinal disease or even blindness [28,31,32]. At an excitation wavelength of 730 nm, TPEF from photoreceptors has been shown to vary over time and to depend on the quantity of available photopigment [26]. Studies suggest that the dominant source contributing to the time-varying TPEF from photoreceptor outer segments is all-trans-retinol, an intermediate component of the visual cycle [33][34][35][36][37][38].…”