Functional Optical Coherence Tomography for Intrinsic Signal Optoretinography: Recent Developments and Deployment Challenges

Abstract

Intrinsic optical signal (IOS) imaging of the retina, also termed as optoretinogram or optoretinography (ORG), promises a non-invasive method for the objective assessment of retinal function. By providing the unparalleled capability to differentiate individual retinal layers, functional optical coherence tomography (OCT) has been actively investigated for intrinsic signal ORG measurements. However, clinical deployment of functional OCT for quantitative ORG is still challenging due to the lack of a standardized imaging protocol and the complication of IOS sources and mechanisms. This article aims to summarize recent developments of functional OCT for ORG measurement, OCT intensity- and phase-based IOS processing. Technical challenges and perspectives of quantitative IOS analysis and ORG interpretations are discussed.

Keywords: functional retinal imaging; intrinsic optical signal imaging; optical coherence tomography; optoretinography; photoreceptor; phototransduction; retina; retinography.

Figure 1

(A) Representative functional OCT system for IOS/ORG imaging. BS, beam splitter; CL, collimation lens; Lenses, L1, L2, L3, L4, L5, and L6; PC, polarization controller; SLD, superluminescent diode. (B) Representative pupil image (B1) and pupillary response (B2). Reprinted with permission from Son et al. (25). (C1) Representative OCT B-scan of the human retina and (C2) reflectance profiles of the central fovea (yellow), parafovea (blue), and perifovea (purple). Yellow, blue, and purple windows in (C1) indicate retinal regions for comparative reflectance profile analysis. The green arrow in (C2) indicates a possible band, i.e., Bruch's membrane. T1–T3 indicate trough positions along the reflectance profiles. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; ISe, inner segment ellipsoid; OS, outer segment; IZ, interdigitation zone; RPE, retinal pigment epithelium; BM, Bruch's membrane. Modified with permission from Yao et al. (56).

Figure 2

Representative OCT intensity-based IOS imaging. (A) Representative OCT image sequence. (B) Corresponding IOS distributions of positive (red) and negative (green) changes. (C) IOS magnitude sequence with 0.1 s time intervals. (D) IOS magnitude sequence with 10 ms time intervals. Reprinted with permission from Son et al. (25).

Figure 3

Representative OCT hyper-reflective band analysis. (A) Deconvolution method. (A1) Depth scattering profiles of the retina of an albino mouse. (A2) Deconvolution analysis reveals that the backscatter band nearest to Bruch's membrane (BrM) on the anterior side comprises two distinct components (question marks are used to indicate that the assignment to structures required confirmation). Reprinted with permission from Zhang et al. (35). (B) A-line signal modeling by summation of seven Gaussian curves. Gray crosses represent the measured data points, and the black line is the summation of the individual model curves (green, blue, red, and gray lines). ELM, external limiting membrane; IS/OS, inner segment/outer segment complex; ROST, rod outer segment tips; RPE, retinal pigment epithelium; CC, choriocapillaris; Cho, choroid. Reprinted with permission from Messner et al. (54).

Figure 4

Phase-resolved OCT imaging for optical path length (OPL) estimation. (A) Optoretinography experimental paradigm. 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) Optoretinography reveals functional activity in cone outer segments. Illumination pattern (three bars) drawn to scale over the line-scan ophthalmoscopic image. (C,D) The spatial map of OPL changes between the ISOS and COST before (C) and after stimulus (D), measured at 20-Hz volume rate. Reprinted with permission from Pandiyan et al. (32).

Figure 5

Phase-resolved OCT imaging for differential-phase mapping (DPM) analysis. (A) A flow chart of amplitude OCT and DPM processing. (B–E) The amplitude-IOS and phase-IOS distribution. (B) Amplitude image sequence. (C) DPM sequence. (D) Amplitude-IOS sequence. (E) Phase-IOS sequence. Reprinted with permission from Ma et al. (37).

Figure 6

Retinal neurovascular coupling and inner retinal IOS response. (A) Representative flattened (A1) OCT B-scan and (A2) spatiotemporal neural-IOS map. (B) Representative flattened (B1) OCTA B-scan and (B2) spatiotemporal hemodynamic-IOS map. Scale bars in (A1,B1) indicate 500 μm. (C) Neural-IOS changes of photoreceptor layer (PL), outer plexiform layer (OPL), inner plexiform layer (IPL), and ganglion cell layer (GCL). (D) Hemodynamic-IOS changes of superficial vascular plexiform (SVP), intermediate capillary plexiform (ICP), and deep capillary plexiform (DCP). (E) Averaged onset times of neural-IOS changes at PL, OPL, IPL, and GCL. (F) Averaged onset times of hemodynamic-IOS changes of SVP, ICP, and DCP. Modified with permission from Son et al. (63). P values for statistical significances are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Photoreceptor outer segment (OS) shrinkage due to light stimulus. (A1) Stimulus-evoked mouse rod OS movement. The yellow window indicates the stimulation area. (A2) OS changes of a mouse rod photoreceptor. In the center of the stimulation region, the length of the OS shrunk, while in the peripheral region, the OS swung toward the center of the stimulation area in the plane perpendicular to the incident stimulus light. Reprinted with permission from Zhao et al. (88). (B1) Representative light microscopic images of an isolated frog rod OS acquired with an interval of 0.5 s. To better show the light-evoked OS shrinkage, the base of the rod OS in each image is aligned horizontally as shown by the black solid line at the bottom. The black-dashed line at the top represents the position of the rod OS tip at time −1 s. Scale bars (in white) represent 5 μm. (B2) Enlarged picture of the white rectangle in (B1). Scale bars (in white) represent 2 μm. (B3) Time course of the averaged rod OS shrinkage in both length and diameter acquired from eight different rod OSs. Colored areas accompanying the curves represent the standard deviations. Shaded area indicates the 1-second stimulation period. Reprinted with permission from Lu et al. (82). (C) Mechanical response of an X. laevis rod to light flashes. The position of a bead sealed against the tip of the rod OS is monitored with optical tweezers. Following a bright flash of 491 nm, equivalent to about 10⁴ photoisomerization [R*], a transient shrinkage is observed. (C1) Bright-field infra-red image, showing a trapped bead in contact with the tip of the rod OS (scale bar, 10 μm). (C2) Detail of the 3D tracking system. (C3) Light-induced shifts in the Z axis of the trapped bead (downward is negative). (C4) Expansion of the time base in C5 to examine the delay between light stimulus and bead movement. (C5) Bead displacement along the direction of the rod OS (shrinkage is negative, and elongation is positive). (C6) Bead displacement in the direction perpendicular to the rod OS axis. Data are representative of mean ± SD of 5 different experiments. Reprinted with permission from Bocchero et al. (89).

Figure 8

Metabolic response of photoreceptor inner segment. (A) IOS M-scan within 100 ms, the white and red arrowheads show the onsets of IS-IOS and OS-IOS, respectively. (B) Average IS-IOS and OS-IOS from six mice. The average OS-IOS showed a biphasic curve. The dark and gray arrowheads show the first rapidly increasing phase and the second gradually increasing phase, respectively. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; ISe, inner segment ellipsoid; IZ, interdigitation zone; RPE, retinal pigment epithelium; Ch, Choroid; IS, photoreceptor inner segment; OS, photoreceptor outer segment. Reprinted with permission from Ma et al. (61).

Figure 9

Subretinal space changes during dark adaptation in the mouse retina. (A) OCT images of light- and dark-adapted retina of a two-month-old C57BL/6J mouse. Color arrows to indicate outer retinal bands: 1st ELM band (yellow), 2nd ISe band (blue), 3rd IZ and OS tip band (red), 4th RPE band (white). (B) A sequence of OCT images obtained every 5 min up to 30 min during dark adaptation. During dark adaptation, ISe intensity reduction rapidly occurred, and the SRS became thinner. In addition, the 3rd outer retinal band (red arrow) faded over time. NFL, nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; ISe, inner segment ellipsoid; IZ, interdigitation zone; RPE, retinal pigment epithelium; CH, choroid. Scale bars: 100 μm. Modified with permission from Kim et al. (65).