In vivo functional imaging of human cone photoreceptors

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.

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

“…As noted in the Introduction, intrinsic optical signals from individual human cone photoreceptors have been measured by a number of groups (10)(11)(12)(13)(14). The advanced state of adaptive-optics OCT methodology for the human eye enables such cone-specific intrinsic optical signals to be characterized with great precision and sensitivity, and rapid advances in measurement and clinical applicability of human photoreceptor optophysiology can certainly be expected (71).…”

“…OCT has been universally adopted in clinical ophthalmology to map human retinal layer structure (2) and is widely used for angiography of the eye (3) and heart (4,5) and for in vivo biopsy of skin and other tissues (6,7). Ocular OCT has also been used in vivo to measure "intrinsic optical signals" from the photoreceptor layer of the retina (8,9), including from individual human cone photoreceptors (10)(11)(12)(13)(14). In no case, however, has the molecular mechanism underlying an intrinsic optical signal been unequivocally identified.…”

Noninvasive imaging of the photoreceptor mosaic response to light stimulation

“…Thus, path length changes can be measured with exquisite precision down to fractions of a wavelength. Jonnal et al (6) elegantly used the photoreceptors themselves as a biological interferometer, building a flood illumination, adaptive optics ophthalmoscope where the two interfering paths were reflections from the proximal and distal ends of the photoreceptor outer segments (Fig. 1).…”

“…1). The resulting oscillations in intensity after a light stimulus suggested that IOSs correlate with a change in the optical path length of the photoreceptors during phototransduction (6).…”

“…This enabled the rapid acquisition of a series of phase-stabilized volumes at rates of hundreds of volumes per second. With this impressive imaging speed, building on the hypothesis by Jonnal et al (6), the authors were able to measure elongation of the outer segments by referencing the phase of the proximal outer segment reflection to the phase of the distal one (Fig. 1).…”