Light-adapted flicker optoretinograms captured with a spatio-temporal optical coherence-tomography (STOC-T) system

Abstract

For many years electroretinography (ERG) has been used for obtaining information about the retinal physiological function. More recently, a new technique called optoretinography (ORG) has been developed. In one form of this technique, the physiological response of retinal photoreceptors to visible light, resulting in a nanometric photoreceptor optical path length change, is measured by phase-sensitive optical coherence tomography (OCT). To date, a limited number of studies with phase-based ORG measured the retinal response to a flickering light stimulation. In this work, we use a spatio-temporal optical coherence tomography (STOC-T) system to capture optoretinograms with a flickering stimulus over a 1.7 × 0.85 mm² area of a light-adapted retina located between the fovea and the optic nerve. We show that we can detect statistically-significant differences in the photoreceptor optical path length (OPL) modulation amplitudes in response to different flicker frequencies and with better signal to noise ratios (SNRs) than for a dark-adapted eye. We also demonstrate the ability to spatially map such response to a patterned stimulus with light stripes flickering at different frequencies, highlighting the prospect of characterizing the spatially-resolved temporal-frequency response of the retina with ORG.

Fig. 1.

Schematic of the STOC-T setup with a path for a patterned flickering stimulus (green dotted lines). DMD – digital micromirror device; DM – dichroic mirror, BS – beam splitter, LED – light-emitting diode, L₁-L₁₂ – achromatic lenses. The multimode fiber provides effective crosstalk reduction. The retinal and reference mirror planes are conjugated to the high-speed and preview camera, while the DMD plane is conjugated to the retina. The reference mirror is slightly tilted so that the reference beam is not blocked by the pick-off (rod) mirror. The inset shows the white light LED spectrum entering the eye pupil.

Fig. 2.

Location and FOV of the photoreceptor layers involved in the ORG signal detection. (a) En face and (b) B-scan structural STOC-T images overlaid on (a) a cSLO fundus image and (b) an SD-OCT B-scan of the subject's retina. (c) Schematic and corresponding STOC-T B-scan showing the location of the IS/OS and COST photoreceptor layers.

Fig. 3.

Schematic illustrating (a) the data acquisition sequence, including synchronized volume acquisition with light stimulation over time; (b) OCT signal processing; and (c-f) multi-step volume alignment procedure – as described in the text.

Fig. 4.

(a) Onset and duration of a uniform (non-patterned) light pulse stimulus. (b) Photoreceptor OPL change over time (average of three separate measurements) for the light pulse stimulus as of (a) (averaged over a 1.70 × 0.85 mm² area of the retina). (c) Map of a spatially-resolved photoreceptor response to a checkerboard-patterned light pulse stimulus 250 ms after the stimulus onset (with details reported in Table 1.) (d) Overlay of the variation of OPL on a STOC-T en-face map showing the photoreceptors layer.

Fig. 5.

Comparison of the ORG signals (e)-(h), averaged over the full FOV, and corresponding spectra (i)-(l) obtained under different illumination conditions, represented in (a)-(d). Plots for a dark-adapted eye before flickering stimulation at 15 Hz (a), (e), (i); light-adapted eye before flickering stimulation at 15 Hz (b), (f), (j); no stimulus (c), (g), (k); and constant non-flickering stimulus (d), (h), (l).

Fig. 6.

Averaged measured filtered ORG response and OPL modulation amplitude spectrum to a 15 Hz stimulus. (a) Averaged, dark-adapted ORG signal, as in Fig. 5(e), after high pass-filtering in solid red. (b) Averaged, light-adapted ORG signal, as in Fig. 5(f) in a black dashed line and after high pass-filtering in solid blue. (c) Modulation amplitude spectrum of the ORG signal in (a). (d) Modulation amplitude spectrum of the ORG signals in (b). The FFT is zero-padded to N = 2048 datapoints. The OPL modulation amplitude spectrum is computed as the amplitude spectrum of the zero-padded signal normalized by half the raw number of time steps (bins).

Fig. 7.

Spectra of OPL modulation amplitude resulting from a spatially-uniform, flickering stimulation with a frequency of (a) 15 Hz, (b) 20 Hz, (c) 25 Hz, and (d) 30 Hz, under light-adapted protocol as in Fig. 5(b). Each plot shows the average (in a solid blue line) and the ± one standard deviation range (in a blue shade) for (a) eight, (b) six, (c) six, and (d) five high pass-filtered ORG measurement spectra. At the bottom of the graphs, the horizontal lines and the asterisks (*) join the pairs of datasets from different flickering frequencies that have statistically different mean peak modulation amplitudes.

Fig. 8.

Measured photoreceptor OPL modulation amplitude in response to a patterned flickering white light stimulation. (a) Measured ORG response to stimulation with a pattern containing 15 Hz flickering stripes. Pre-flicker photon flux of $13\cdot {10}^2\text{photons}/\text{ms}/\mu {\text{m}}^2$ , flickering peak photon flux I₀ =  $13\cdot {10}^2\text{photons}/\text{ms}/\mu {\text{m}}^2$ . (b) Measured ORG response to stimulation with a pattern containing alternately 15 Hz and 18 Hz flickering stripes (see Visualization 1). Pre-flicker photon flux of $12\cdot {10}^2\text{photons}/\text{ms}/\mu {\text{m}}^2$ flickering peak photon flux I₀ =  $12\cdot {10}^2\text{photons}/\text{ms}/\mu {\text{m}}^2$ .