Optoretinography with actively stabilized adaptive optics optical coherence tomography

Abstract

Optoretinography (ORG) is the optical measurement of changes in the retina in response to light stimulation. Adaptive optics optical coherence tomography (AOOCT) records photoreceptor ORGs by measuring the physical changes in their outer segment lengths in response to light stimulation. The main difficulty in recording these nanometer-scale changes is constant eye motion. Typically, fast volume acquisitions are used with offline spatial registration to compensate for the effect of eye motion. Here, we present an alternate solution whereby an adaptive optics scanning light ophthalmoscope (AOSLO) is used to measure the eye motion and actively guide the AOOCT beam to compensate for eye motion in real time. This system's cellular-scale tracking offers unparalleled control over scanning raster size and shape, allowing for high-speed (up to 100 kHz) ORG acquisition from targeted locations. We validate the method by comparing cone classifications against those made with an established ORG approach.

Fig. 1.

Strip based eye tracking. As each AOSLO frame is scanned, it is compared, strip-by-strip to a reference frame, and the OCT scanners offsets are adjusted in real time, based on the registration. The reporting rate for eye position is the AOSLO frame rate multiplied by the number of strips per frame. In the current system the rate is 30 (fps) × 32 (strips per frame) = 960 Hz.

Fig. 2.

Schematic of the combined AOSLO-AOOCT system. Red trace indicates the AOSLO optical path, blue trace indicates the AOOCT optical path and green indicates where the two optical paths are common. More details on the system are described in other publications [43,44].

Fig. 3.

AOOCT scanning location and sizes relative to the AOSLO reference frame. The black box denotes the structural scan and the blue, red, and green boxes denote the repeated A-, B-, and C-mode ORG scans respectively.

Fig. 4.

a) ORG scan of single cell using C-mode with a small field of view and eye tracking. b) AOOCT structural (top) and ORG scans (bottom) in C-mode with and c) without active eye motion correction. The green box in the ORG scans denote the size and shape of a single ORG scan, and corresponds to the green box in the structural scan. All scale bars are 10 arcmin. A video sequence of the stabilized ORG frames can be found in Visualization 1

Fig. 5.

a1) Representative A-mode A-scan time series from Subject 20255. Red and blue lines indicate the approximate location of the IS/OS and COST respectively. a2) Averaged A-mode ORG traces from Subject 10003 (orange) and 20255 (blue). The transparent traces are raw data, and the solid traces have been smoothed via moving window average of 3000 samples (or 0.03s) to better show the contractile response. b1) Representative B-mode en face time series from subject 20255 and b2) B-mode averaged trace. The trace was averaged both across multiple trials and spatially across a B-scan. Again, the transparent traces are raw data and the solid traces are smoothed with a moving window average of 10 samples (or 0.04s). Vertical green lines indicate 543 nm stimulus onset.

Fig. 6.

a) ORG traces of cones in Subject 10003’s classified region (1.7 degrees temporal), where the colors represent our collaborator’s assigned cone classes: red for L cones, green for M cones, and blue for S cones. b) Comparison of experimental class assignments and collaborator assignments. c) ORG traces from an unclassified region of Subject 20255 (2.9 degrees temporal). Vertical red line indicates 650 nm stimulus onset. d) Retinal images of both subjects with cone class labels overlaid. Scale bars are 10 arcmin.

Fig. 7.

a) OCT en face image of Subject 10003. Three classified cones are colored based on collaborator cone subtype assignments. Scale bar is 5 arcmin. b) Resulting ORG traces. Colors are again based on collaborator assignments. The transparent traces are raw data, and the solid traces have been smoothed via moving window average of 3000 samples (or 0.03s). Vertical red line indicates 650 nm stimulus onset.