Abstract
We evaluate a novel non-invasive optical technique for observing fast physiological processes, in particular phototransduction, in single photoreceptor cells in the living human eye. The method takes advantage of the interference of multiple reflections within the outer segments (OS) of cones. This self-interference phenomenon is highly sensitive to phase changes such as those caused by variations in refractive index and scatter within the photoreceptor cell. A high-speed (192 Hz) flood-illumination retina camera equipped with adaptive optics (AO) is used to observe individual photoreceptors, and to monitor changes in their reflectance in response to visible stimuli ("scintillation"). AO and high frame rates are necessary for resolving individual cones and their fast temporal dynamics, respectively. Scintillation initiates within 5 to 10 ms after the onset of the stimulus flash, lasts 300 to 400 ms, is observed at visible and near-infrared (NIR) wavelengths, and is highly sensitive to the coherence length of the imaging light source. To our knowledge this is the first demonstration of in vivo optical imaging of the fast physiological processes that accompany phototransduction in individual photoreceptors.
Highlights
Human vision starts when photoreceptors collect and respond to light, a complex biochemical cascade of events referred to as phototransduction
Each set of ten cones exhibited the highest time-RMS on their respective halves of the video frame. Visual comparison of both the full field video and the enlarged individual cone videos clearly shows that scintillation is confined to the stimulated portion of the retina and occurs only after onset of stimulus
Three experiments were conducted to (1) test the utility of a high-speed adaptive optics (AO) flood-illumination camera for cellular retinal imaging and (2) explore the optical behavior of individual cone photoreceptors when exposed to light stimuli
Summary
Human vision starts when photoreceptors collect and respond to light, a complex biochemical cascade of events referred to as phototransduction. The phototransduction process has been extensively studied in vitro, resulting in detailed models of the biochemical cascade (see, for example, [1]) Techniques to assess these processes in vivo, are limited. Direct observation in the living human eye has been confined to the initial photon absorption kinetics (measured by photopigment densitometry [2]) and the final membrane hyperpolarization (measured by electrophysiology [3,4]). While these techniques have allowed macroscopic investigation of large patches of retina, they have not allowed probing of the function of individual photoreceptors. Since the silicon of the CCD is more transparent at 915 nm than it is at 835 nm or 670 nm, the etaloning was predicted to be less significant at the latter wavelengths; contrast of the fringes at 835nm was measured to be approximately 10%, and negligible at 670 nm
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