The optoretinogram reveals the primary steps of phototransduction in the living human eye

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Science Advances  09 Sep 2020:
Vol. 6, no. 37, eabc1124
DOI: 10.1126/sciadv.abc1124
  • Fig. 1 Optoretinography experimental paradigm.

    (A) High-resolution volumetric imaging using an a line-scan retinal camera. Human retina was visualized using a multimodal camera consisting of fast volumetric imaging with line scan OCT, en face LSO imaging, and a light stimulus in Maxwellian view. A three-dimensional (3D) OCT volume with AO allows resolving the cone mosaic in an en face projection and the outer retinal layers in an axial profile corresponding to the ISOS and COST. Stimulus (528 ± 20 nm, green)–driven changes in a cone photoreceptor are accessible by computing the time-varying phase difference between the proximal and distal OCT reflections encasing the outer segment. (B) Modes of acquisition and processing. In both normal and ultrafast modes, a stimulus with pulse width (τo) and delay (τd) relative to the start of OCT recording illuminated the retina. The OCT volumes (V) were referenced in time to the stimulus onset and ranged from V−τd to Vn. In the normal mode, the phase difference between ISOS and COST was computed for each volume (ϕvol) and converted to OPL to represent the optical change in cone outer segments following light stimulation. The volume rate (120 to 324 Hz) defined the temporal resolution of the response in the normal mode. In the ultrafast mode, the phase difference was computed for each B-scan within a volume (ϕB-scanvol) and the B-scan rate (16 kHz) defined the temporal resolution.

  • Fig. 2 Optoretinography reveals functional activity in cone outer segments across different spatial scales.

    (A) Illumination pattern (three bars) drawn to scale over the LSO image. (B and C) The spatial map of OPL change between the ISOS and COST before (B) and after stimulus (C), measured at 20-Hz volume rate. (D) Rectangles over an LSO image represent the areas over which averages were obtained to plot the ORGs: 0.27°2 (yellow), 0.20°2 (gray), 0.14°2 (red), and 0.07°2 encompassing ≈10 cones (violet). (E) Repeatability of the response: single ORGs (gray, dashed) and their mean (solid black) for six repeat trials, where phase responses were averaged over 0.27°2 for 17.9% bleach. (F) Spatial averaging: ORGs over different areas color-coded according to the rectangles in (D). (G) Maximum intensity projection at COST layer with AO-OCT reveals individual cone photoreceptors. (H) ORGs for a subset of single cones in (G) demonstrating the response in each cone for 0.3% S- cone bleach and 29.7% average L and M cone bleach. The magnified view near stimulus onset shows a negligible early response in putative S-cones (blue) compared to L/M cones (orange). (I) ORG early and late response amplitudes for each cone in (G). (J) Histogram of the ORG early and late response magnitude, computed as the Euclidean distance from origin of each data point in (I). The two-component Gaussian mixture model (black dotted line) and its component Gaussians are used to distinguish S-cones (blue fit) from L/M cones (orange fit). The vertical dotted line marks t = 0 in (E), (F), and (H), indicating the rising edge of stimulus onset. (A) to (F) are obtained without AO, with 4-mm imaging pupil, at 120-Hz volume rate. (G) and (H) are obtained with AO, for 6-mm imaging pupil, at 162-Hz volume rate. The stimulus wavelength for all plots is 528 ± 20 nm.

  • Fig. 3 Stimulus strength dependence of the ORG.

    (A) The early and late response components of the ORG are shown as a function of stimulus strength, expressed here as photon density (photons per square micrometer) and bleach percent. A magnified view of the early response at higher temporal resolution is plotted in (D). The maximum OPL change in the late and early responses are shown in (B) and (E), respectively, with their corresponding best fits (solid line in blue and red) for two subjects. Four individual measurements were averaged for all data shown here in (A) and (D), and error bars denote ±1 SD from the mean. OPL changes are averaged spatially over 0.27°2. (C) shows the slope of the swelling in the late response for two subjects and its best fit. (F) Early response recorded at a high temporal resolution reveals that the onset latency is minimal with respect to the flash onset. Eight individual measurements are averaged in (F). The vertical dotted marks t = 0 in (A) and (F) indicate the rising edge of stimulus flash. All data in these figures were collected without AO and with a 4-mm pupil. Volume rates for (A) and (B) is 120 Hz, (C) to (E) is 324 Hz, and (F) is 32 Hz. The stimulus wavelength for all plots is 528 ± 20 nm.

  • Fig. 4 Mechanism of cone outer segment contraction.

    (A) Repulsion of charges in the hyperpolarized disc membrane leads to its expansion, which results in flattening of the disks due to volume conservation. The illustrations of the cone outer segments shown are not to scale. (B) Logarithmic model fit to the maximum amplitude of the early response shown in Fig. 3E. (C) Model fit to the time to peak of the early response.

  • Fig. 5 Optoretinography schematic and representative retinal images.

    (A) Block diagram of optoretinography system showing key components and features (details in Materials and Methods). AO-LSO (B) and AO-OCT images (C) of cone photoreceptors at 7° eccentricity. The images are registered and field flattened to compensate for the Gaussian intensity distribution along the line dimension. SLD, Superluminescent diode; CMOS, complementary metal-oxide semiconductor.

  • Table 1 Summary of stimulus and imaging parameters for optoretinography.

    Stimulus parametersImaging parameters
    Pulse width
    τ0 (ms)
    Delay (τd)
    Spatial size
    Bleach* (%)Speed (Hz)
    B-scan/vol rate
    size (mm)
    Field of
    view scan x
    line (°)
    No. of volsNo. of
    ORG early
    520 vols37.50.4 – 18.516200/32440.6 × 1.41624
    ORG late
    3 – 7010 vols37.51.2 – 48.48400/12040.6 × 1.41204
    ORG ultrafast
    510 vols and
    100 B-scans
    37.53.0 – 29.716000/3241.7 × 1.4258
    ORG early and
    with AO
    510 vols37.529.716200/16260.8 × 1.48012

    *Bleach percent for average of L/M cones. Vols, volumes.

    Supplementary Materials

    • Supplementary Materials

      The optoretinogram reveals the primary steps of phototransduction in the living human eye

      Vimal Prabhu Pandiyan, Aiden Maloney-Bertelli, James A. Kuchenbecker, Kevin C. Boyle, Tong Ling, Zhijie Charles Chen, B. Hyle Park, Austin Roorda, Daniel Palanker, Ramkumar Sabesan

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      • Supplementary Text
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      • References

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