On the basis of these findings, we suggest that ascription of the outer retinal band 2 to the inner segment ellipsoid is unjustified, because the ellipsoid is both too thick and proximally located to produce the band.

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.

Abstract:We report the first observations of the three-dimensional morphology of cone photoreceptors in the living human retina. Images were acquired with a high-speed adaptive optics (AO) spectral-domain optical coherence tomography (SD-OCT) camera. The AO system consisted of a Shack-Hartmann wavefront sensor and bimorph mirror (AOptix) that measured and corrected the ocular and system aberrations at a closed-loop rate of 12 Hz. The bimorph mirror was positioned between the XY mechanical scanners and the subject's eye. The SD-OCT system consisted of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquired 75,000 A-scans/s. This rate is more than two times faster than that previously reported. Retinal motion artifacts were minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera sensitivity was sufficient to detect reflections from all major retinal layers. The regular distribution of bright spots observed within C-scans at the inner segment / outer segment (IS/OS) junctions and at the posterior tips of the OS were found to be highly correlated with one another and with the expected cone spacing. No correlation was found between the posterior tips of the OS and the other retinal layers examined, including the retinal pigment epithelium.

With the use of adaptive optics (AO), high-resolution microscopic imaging of living human retina in the single cell level has been achieved. In an adaptive optics confocal scanning laser ophthalmoscope (AOSLO) system, with a small field size (about 1 degree, 280 µm), the motion of the eye severely affects the stabilization of the real-time video images and results in significant distortions of the retina images. In this paper, Scale-Invariant Feature Transform (SIFT) is used to abstract stable point features from the retina images. Kanade-Lucas-Tomasi(KLT) algorithm is applied to track the features. With the tracked features, the image distortion in each frame is removed by the second-order polynomial transformation, and 10 successive frames are co-added to enhance the image quality. Features of special interest in an image can also be selected manually and tracked by KLT. A point on a cone is selected manually, and the cone is tracked from frame to frame.Small table-top adaptive optical systems for human retinal imaging," Proc. SPIE 4825, 99-105 (2002).

In vertebrate eyes, vision begins when the photoreceptor's outer segment absorbs photons and generates a neural signal destined for the brain. The extreme optical and metabolic demands of this process of phototransduction necessitate continual renewal of the outer segment. Outer segment renewal has been long studied in post-mortem rods using autoradiography, but has been observed neither in living photoreceptors nor directly in cones. Using adaptive optics, which permits the resolution of cones, and temporally coherent illumination, which transforms the outer segment into a “biological interferometer,” we observed cone renewal in three subjects, manifesting as elongation of the cone outer segment, with rates ranging from 93 to 113 nm/hour (2.2 to 2.7 μm/day). In one subject we observed renewal occurring over 24 hours, with small but significant changes in renewal rate over the day. We determined that this novel method is sensitive to changes in outer segment length of 139 nm, more than 20 times better than the axial resolution of ultra-high resolution optical coherence tomography, the best existing method for depth imaging of the living retina.