Optoretinogram: optical measurement of human cone and rod photoreceptor responses to light

Abstract

Noninvasive, objective measurement of rod function is as significant as that of cone function, and for retinal diseases such as retinitis pigmentosa and age-related macular degeneration, rod function may be a more sensitive biomarker of disease progression and efficacy of treatment than cone function. Functional imaging of single human rod photoreceptors, however, has proven difficult because their small size and rapid functional response pose challenges for the resolution and speed of the imaging system. Here, we describe light-evoked, functional responses of human rods and cones, measured noninvasively using a synchronized adaptive optics optical coherence tomography (OCT) and scanning light ophthalmoscopy (SLO) system. The higher lateral resolution of the SLO images made it possible to confirm the identity of rods in the corresponding OCT volumes.

Fig. 1.

Example of simultaneously acquired AO-SLO-OCT images at 6° temporal to the fovea. Rods are not as well resolved in the OCT en face projection, as shown in (A), as they are in the SLO image, as shown in (B). The scale bar is 10 μm.

Fig. 2.

(A) and (B) show the OCT en face projections acquired 6° temporal to the fovea in two trials of different stimulus intensities, while (C) and (D) show plots of the corresponding elongation of select rods and cones in the field, and (E) and (F) show the corresponding averaged responses of 10–20 cones and rods. In (A), (C), and (E), the flash bleached 0.2% and 0.05% of L/M photopigment and rod pigment, respectively, while the brighter flash in (B), (D), and (F) bleached 4.0% and 1.0%, respectively. No cone elongation is visible in response to the dimmer flash in (C) and (E), whereas a clear rod response is visible. In response to the brighter flash in (D) and (F), both rods and cones elongate, with the elongation of rods having several times higher amplitude. The averaged cone and rod responses to a still brighter flash, which bleached 15% and 3.75%, are shown in (G), where the cone response appears to have a higher initial slope and the rod response appears to saturate. Together these results are consistent with key fundamental differences between the cells—that cone responses are faster but less sensitive than those of rods. M-scans of a single cone and rod from the field are shown in (H), along with plots of their time-averaged axial reflectance profiles. The longer OS of the rod and corresponding distal displacement of its OS tip are clearly visible. This morphological difference aids in classification of the cells, and also guards against cross talk of their responses due to lateral blur. Colors of arrows in (A) and (B) correspond to line colors in (C) and (D), and shaded regions in (E)–(G) represent the standard error of the mean (±SEM). The scale bar is 10 μm.

Fig. 3.

(A) shows the averaged response of 30–50 rods for each of eight flash intensities in subject 1. (B) shows the same for each of the four flash intensities in subject 2. In each plot, the dashed line and shaded area show the average response and ±SEM. The solid lines are the result of local smoothing by rolling average. Various characteristics of the response curves—such as initial velocity and maximum excursion—appear qualitatively related to bleaching percentage. (C) shows a plot of elongation velocity, averaged over 1.5 s post-flash, as a function of bleaching percentage for both subjects; each marker on the plot represents one of the curves in (A) and (B). These were fit with two models, a log-linear model and a sigmoidal Michaelis–Menten function, both of which fit the data well (R²≥0.89).

Fig. 4.

M-scans of rods [shown in (A)] and cones [shown in (B)] for photopigment bleaching percentages of 4% and 15%, respectively, reveal similar changes in the apparent axial morphology of the cells. As shown by red arrows, appearance of an extra band between IS/OS and ROST (COST) was observed in most of the rods (cones). The reflectivity of this extra band and also its axial distance from IS/OS seem to be proportional to the bleaching light intensity. The yellow arrow indicates changes observed in the RPE and subretinal space.