Interferometric imaging of the reversible rhodopsin dynamics in the living rat eye

Domain: physics
Published: 2025-11-28
Editorial outcome: minor_revision

Abstract
Vision begins with conformational changes in photopigments. The associated electrical signature, called an early receptor potential (ERP), in rods is limited to contribution of a small fraction of rhodopsin embedded in plasma membrane. Optoretinography (ORG), using phase-sensitive optical coherence tomography, detects nanoscale deformations of retinal cells associated with physiological processes. In previous ORG studies, focused primarily on cones, deformation related to ERP was largely obscured by osmotic swelling and long stimuli. Here, we demonstrate a robust electromechanical signature of photoisomerization in rods. A green flash induces a sub-millisecond contraction of the outer segments by hundreds of nanometers, while a subsequent UV flash reverts the activated molecules, producing an opposite response of similar magnitude. ORG surpasses the sensitivity of electrical methods by integrating the response across all the discs in rod outer segments and it opens the door to fundamental studies of visual transduction in-vivo and to more specific clinical diagnosis.

Introduction
Vision begins with the absorption of photons in photoreceptors, triggering photoisomerization of photopigments and subsequent phototransduction, ultimately hyperpolarizing the cells and modulating their neurotransmitter release1. After photon capture, rhodopsin undergoes a series of isomerization intermediates, including lumirhodopsin and metarhodopsin I (Meta I), before reaching the biochemically active metarhodopsin II (Meta II) state2,3, a process involving charge transfer across the membrane, primarily in the outer segment discs4–6. Patch clamp electrophysiological recordings in photoreceptors, a very invasive technique applicable only ex vivo, revealed a rapid electrical signal, called the early receptor potential (ERP)6–8: a brief depolarizing phase (R1) corresponding to the formation of Meta I, and a dominant hyperpolarizing phase (R2) reflecting the conversion from Meta I to Meta II8. Previous electrical recordings in living rat eyes demonstrated that a 400-nm flash delivered to photoreceptors fully bleached by green light can elicit a rapid electrical signal of opposite polarity, indicating that near-UV photons can revert the activated rhodopsin into another isoform9, subsequently shown to be Metarhodopsin III (Meta III), which can again absorb green photons10.

In vivo observation of these processes is critical for understanding the biophysics of vision in real physiological conditions and for more specific clinical diagnosis of multiple forms of photoreceptors’ dysfunction. Indeed, a reduction in ERP amplitude has been noted in electroretinography (ERG) recordings from patients with diabetic retinopathy and retinitis pigmentosa11. The ERP originates from charge transfer across the cell membrane5,6. Even though rods constitute the vast majority of photoreceptors in most mammalian retinas12, such electrical recordings from rods are more challenging than from cones due to the distinct membranous organization of their outer segments. Specifically, most of rhodopsin is embedded in the sealed disc membranes, which are isolated and electrically decoupled from the plasma membrane of the outer segment (Fig. 4A)3,13. In contrast, cone discs are direct infoldings of the plasma membrane itself14. As a result, rod signals are overshadowed by cone responses in primate and human retinas15,16.

Optical coherence tomography (OCT) is a powerful imaging modality in biomedical optics that enables non-invasive, depth-resolved imaging with micron-level resolution17. While the amplitude component of OCT signals reveals microstructural features, phase-sensitive measurements allow motion detection with nanometer-scale sensitivity18,19. In particular, by monitoring the phase difference between two retinal layers20, phase-sensitive OCT has detected deformations in photoreceptors associated with metabolic events, such as photoisomerization and phototransduction21–29. This technique, termed optoretinography (ORG), enabled the observation of a rapid (few milliseconds) contraction and slow (hundreds of milliseconds) expansion of the cone outer segments (COS) immediately following a light stimulus21,23,24. Contractile deformations were attributed to electromechanical coupling30—changes in the surface tension associated with the changes of transmembrane voltage induced by charge transfer across the membrane. In contrast, the slower expansion of COS was linked to osmotic influx of water, swelling of the cone opsin and disc membrane27,31, and conformational changes in phosphodiesterase (PDE6) during phototransduction29. Typically, the swelling in cones obscures the rapid contractile signal related to ERP, and relatively long light stimuli used in earlier ORG studies21,23,24 could not resolve the sub-ms dynamics of the rhodopsin isomerization2,3,9.

In this study, we investigated ORG signatures associated with rhodopsin dynamics in the living rat eye, where ~99% of photoreceptors are rods32, making it an ideal model for studying rod ORG signals. By employing ultrafast (10-kHz B-scan rate) OCT recordings and microsecond-scale light stimuli, we observe a sub-ms fast contraction of the rod outer segments (ROS) by hundreds of nanometers that persisted for hundreds of milliseconds. About a thousand ROS disc membranes collectively contributed to this robust electromechanical effect, surpassing the sensitivity of electrical measurements. Furthermore, a UV (385nm) flash delivered after extensive bleaching by green (520nm) light reversed the contraction and enabled further absorption of green photons, indicating a transition from Meta II to Meta III. This imaging method with microsecond-scale temporal resolution opens the door to fundamental studies of visual transduction in vivo and to more specific clinical diagnosis of the photoreceptors’ dysfunction, with a possibility of single-cell spatial resolution.

Results
Dynamics of the ROS contraction
To assess the dynamics of the ROS contraction, we applied green (520nm) flashes to retinas of wild-type rats while acquiring repeated B-scans at a 10 kHz frame rate using a custom-built ultrafast line-scan OCT system. To investigate the impact of pulse duration on the waveform of the rapid contractile response (Fig. 1), we employed pulse durations of 50, 100, 200, 500 μs, and 1 ms (see Fig. S4 for temporal profiles) and adjusted the power to maintain a consistent pulse energy of −50.6±0.2 μJ, measured in front of the cornea. Considering a transmittance of 0.77 through the anterior segment of the eye at 520nm33,34 and an illumination area on the retina of 1.88mm2, this corresponds to approximately 5.4×107 photons/μm2 incident on the retina. We also explored the stimulus strength dependence of the rapid contraction by fixing the pulse duration at 100 μs and adjusting the flash energy in approximately two-fold increments (Fig. 2).

Changes in the phase difference between two retinal bands reflect variations in the optical path length (ΔOPL) between them. As illustrated in Fig. 1A, to assess the light-evoked response of the ROS, we computed the phase difference between the inner segment/outer segment (IS/OS) junction and the rod outer segment tips (ROST, the top of the thick hyperreflective band consisting of ROST and the retinal pigment epithelium28,35). The ΔOPL traces presented in Fig. 1B for each pulse duration were extracted from ROS and averaged across 4–5 rats. Since similar photon densities were delivered to the retina, the resulting rapid contraction amplitudes of the outer segments were comparable, although response to the 1-ms flash was noticeably slower and its contraction amplitude was slightly larger than those with shorter pulses. This is likely due to a more pronounced photoreversal effect of shorter pulses, resulting in fewer activated rhodopsins (see section Modeling the ROS contraction). Amplitudes of the rapid contraction were extracted by averaging ΔOPL at the first 3–4 ms temporal window after the stimulus onset, and their statistical distributions are shown in Fig. 1E.

In stark contrast to the fast recovery (<20ms) from the rapid contraction observed in COS responses24, the ROS remained in a contracted state throughout the 400-ms acquisition window. As can be seen in Fig. 1C, the contraction process begins almost immediately after the flash onset, but continues for about 3 ms, even with the 50-μs flash. The contraction rate, shown in Fig. 1D, was calculated by taking a time derivative of each trace. Latency of the maximum contraction rate was extracted and compared to pulse durations in Fig. 1F. Interestingly, for flashes shorter than 0.5 ms, latency of the maximum contraction rate remains almost the same—about 0.45 ms, and it starts increasing for longer pulses.

While a short, intense flash revealed the impulse response of the rapid contraction process, its strength dependence provides additional insights into the underlying mechanisms. Figure 2A displays the ΔOPL traces in response to 100-μs flashes with varying energy levels. As expected, amplitude of the rapid contraction increases with incident photon density. With the lowest pulse energy, OPL significantly recovered during the 400 ms after the flash, while at higher energy levels, contraction remained steady and even slowly increased over time. Dynamics of the rapid contraction within the first 3 ms is shown in Fig. 2B. The contraction amplitudes, determined by the same procedure as in Fig. 1, are shown in Fig. 2D, revealing a saturating trend. Latency of the maximum contraction rate, shown in Fig. 2C, E, decreases with increasing pulse energy from about 0.55 ms and saturates at around 0.45 ms.

Modeling the ROS contraction
Early photoproducts, up to Meta I, can absorb a second photon and restore either rhodopsin or isorhodopsin, a process called photoreversal36–38. Therefore, if a large number of photons are delivered while a substantial portion of rhodopsin isoforms have not yet decayed to Meta II, photoreversal of the rhodopsin bleaching cannot be neglected. Given that the flash durations in our experiments are comparable to the thermal decay time constant from Meta I to Meta II (a few hundred microseconds in rodents5,9), we considered all critical light-driven and thermal processes involved in flash photolysis to determine the bleach levels (Fig. 3A and Supplementary Information: Bleach level calculation). The calculations showed that rapid contraction amplitude ΔOPL increased logarithmically with the estimated bleach levels (Fig. 3B).

The rapid contraction measured in COS was previously explained using the voltage-dependent membrane tension model30. In brief, the charge shift during the R2 phase of the ERP leads to hyperpolarization of the disc membranes. Increase of charge density in the Debye layer increases the repulsion of ions39–41, resulting in lateral stretching of the disc membrane. Due to conservation of the discs’ volume during millisecond-scale dynamics, this lateral expansion leads to the axial contraction of the discs. The cumulative contraction of approximately a thousand discs results in the contraction of the photoreceptor OS by tens or even hundreds of nanometers. As detailed in Supplementary Information, these considerations yield a good match between the modeled and measured contraction amplitude across varying bleach levels (see Fig. S1). At the best fit, we obtained an initial membrane tension τ0 of 0.59 μNm−1 and a bending modulus κc = 0.23 × 10−19 Nm—within the ranges of the membrane surface tension and bending modulus in the literature: 0.1–1 μNm−1 and 0.5–2 × 10−19 Nm30, respectively.

However, this quasistatic model failed to capture the latency of the contraction (see blue curves in Fig. 4B, C), indicating a potential involvement of viscous effects. One factor that could dampen the response, as proposed in our previous study30, is the viscous force exerted by surrounding fluid when individual discs expand laterally. Another mechanism, proposed in this study, arises from the friction when discs move axially in the cytoplasm. Axial shrinkage of each disc results in stretching of the spacers connecting adjacent discs31,42,43, which then causes discs and ROST to move axially. The viscous force induced by the surrounding cytoplasm during this axial movement dampens the contraction process.

A combined model considering both, the lateral and axial viscous and elastic forces applied to each disc, enabled a better fit to experimental results (Fig. 4 and Supplementary Information: Computational Model for ROS Contraction). As shown in Fig. 4B, C, using the 1-ms pulse measurement as an example, the model provides a better fit for the temporal evolution of ΔOPL than its quasistatic approximation. The best fit yielded damping coefficients of 4.0 × 105 Nsm−3 for the lateral expansion of disc membranes and 4.4 × 104 Nsm−3 for the axial movement of the discs. These damping coefficients correspond to the linear damping behavior caused by cytoplasmic sheets of approximately 10 nm and 100 nm in thickness, respectively44, which is reasonable for the membranous structure of ROS42,43. As shown in Fig. 4D, E, the viscoelastic model accurately predicts the latency of the maximum contraction for various pulse durations and energy levels, whereas the quasistatic model exhibits much faster dynamics (blue curves in Fig. 4B, C).

Reversed isomerization with UV flash
Previous studies have demonstrated that an near-UV flash delivered to Meta II can elicit an electrical signal of opposite polarity compared to typical ERP9,45. While the hyperpolarizing phase of the ERP is associated with the transition from Meta I to Meta II, near-UV photons can induce a reversed conformational change from Meta II to Meta III10. To assess the ORG response related to this process, a 1-ms flash at 385 nm was applied after the rhodopsin bleaching by a 1-ms green flash. Pulse energy of the green flash was sufficiently high (340 μJ, 3.6 × 108 photons/μm2 on the retina) to bleach nearly all rhodopsin, as validated by the very small residual response to the second green flash 200 ms later (Fig. 5A). At this pulse energy, thermal effects were still negligible compared to ORG signals (see “Discussion”).

UV flashes were delivered 100 ms after each green pulse at UV energy levels of 30, 70, 112, and 224 μJ, measured in front of the cornea. As shown in Fig. 5B, the first UV flash (1 ms, 112 μJ, 7.5 × 107 photons/μm2 on the retina), applied to an extensively bleached retina, elicited a pronounced elongation of the ROS (with ~10 ms time constant), suggesting a reversed response relative to the contraction induced by the first green flash. The second green flash, applied 100 ms after the UV pulse, induced stronger contraction compared to the control (Fig. 5A), implying partial recovery of photopigment, restoring its ability to respond to green photons. The final UV flash elicited another elongation, similar in amplitude to the first UV pulse.

Amplitude of the elongation produced by the first UV flash exhibited a saturating trend with pulse energy, as shown in Fig. 5C. As anticipated, higher energies of the first UV flash increased the contraction amplitude for the second green flash (Fig. 5D) since more photopigments sensitive to green photons were regenerated by more intense UV light, although this effect saturated beyond 112 μJ. Amplitude of the elongation induced by the final UV flash followed a pattern similar to that of the first one (Fig. 5E).

Remarkably, the UV flash evoked different ORG responses in light-adapted (Fig. 5B) and dark-adapted retinas (Fig. 5F). When delivered to a dark-adapted retina without a preceding green pulse, the UV flash elicited a rapid contraction by 200 nm (Fig. 5F), similar to the response observed with a green flash. After the rapid contraction, however, the ROS briefly expanded by a few tens of nm before undergoing a secondary, slow contraction. The second UV flash, 200 ms later, produced a small additional contraction (like the second green flash in Fig. 5A), but followed by a more pronounced elongation (~10 ms time constant), lasting for hundreds of ms.

Methods
Phase-sensitive OCT imaging
The high-speed line-scan spectral-domain OCT was assembled according to the optical layout shown in Fig. 7. The OCT beam from the supercontinuum laser (FIR-9, NKT Photonics, Denmark) was collimated using lens CL1 (AC254-060-B, Thorlabs) and filtered through two spectral filters (Semrock: FF01-776/LP-25, Thorlabs: FESH0900), producing a bandwidth with a full width at half maximum (FWHM) of about 120 nm centered at 840 nm.

The beam then passes through a cylindrical lens (CYL1), which forms a line image along the y-axis at the focal plane. Following optical conjugation through the afocal telescope L0-L1, the beam was divided into reference and sample arms using a non-polarizing beamsplitter (BSS11, Thorlabs) with a 30:70 (R:T) ratio. Two more afocal telescopes in the sample arm, L2-L3 and L4-L5, conjugate the one-dimensional galvo scanning (GS) mirror (8310K Series Galvanometer, Cambridge Technology) to the system pupil plane. The lens L5 and the animal fixation stage were mounted on a Badal platform, such that translating the platform alters the vergence of the beam entering the eye, enabling compensation for subject-induced defocus. The incident power on the cornea was 2.8 mW. The irradiated line length on the retina was estimated to be 330 μm, and the diffraction-limited resolution (FWHM) was estimated to be 3.6 μm. The elliptical reference beam after the beamsplitter was re-collimated by a cylindrical lens (CYL1R), followed by an afocal telescope (L1R-L2R). A prism pair (#43-649, Littrow Dispersion Prism, Edmund Optics) was used to balance the dispersion mismatch between the reference and sample arms. In the detection path, an anamorphic telescope (L2-CYL2-CYL3) was used to conjugate image planes for optimizing the spatial and spectral resolution simultaneously. An adjustable slit was placed at the image plane to minimize stray light or back-reflections from the contact lens.

The backscattered sample beam and reflected reference beam were diffracted by a 600 l/mm grating (WP-600/840-35×45, Wasatch Photonics), and the image was finally conjugated to the detector of the high-speed camera (Phantom v641) via the telescope (L6-L7), yielding a raw image size of 768×512 pixels (spectral × spatial). In this arrangement, each frame of the camera generated a B-scan of the retina. Data acquisition and real-time processing were implemented in a desktop client application developed using C# on the Windows Presentation Foundation (WPF) framework (XAML 1.0) in Visual Studio 16.0, utilizing the National Instruments API version 19.0. The line-scan ophthalmoscope (LSO) camera (spL4096-140km, Basler) was aligned to be parfocal with the OCT camera. LSO imaging was used for finding a focal plane of the retina, while adjusting the Badal platform, prior to OCT imaging.

Optical paths of retinal stimulation
The green flash path was coupled with the OCT illumination path using a dichroic mirror (NFD01-532-25×36, Semrock). The output of the green laser (520 nm semiconductor laser, Civillaser) was coupled into an optical fiber (MHP200L02 - Ø200 μm Core, 0.22 NA, Thorlabs) using an objective lens LG1 (Fig. 7C). The output of the fiber was optically conjugated to the system pupil plane via an afocal telescope (LG2-L5), which was illuminated in a standard area on the eye, the model in 64. For UV stimulus, the collimated output of a high-power LED (385 nm, SOLIS-385C, Thorlabs) was focused by LUV1 and illuminated a 2.5-mm pinhole (P2500K, Thorlabs) at an intermediate image plane, which was conjugated to the system pupil plane using an afocal telescope (LUV2-L5). At the pupil plane, the beam size was about 2 mm, ensuring that the beam will not be cropped by the rat pupil on its way to the retina. The illuminated area on the retina was estimated to be 1.56 mm2. The UV light was coaxially aligned with the OCT beam using DCM2 (FF495-Di03-25×36, Semrock).

Animal preparation
All experimental procedures were approved by the Stanford Administrative Panel on Laboratory Animal Care and conducted in accordance with the institutional guidelines and conformed to the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology (ARVO). Long Evans rats (male=8, female=7, age: 11–17 weeks) were used, with ad libitum. For all the ORG experiments, animals were dark-adapted more than 12 h overnight. Animals were anesthetized with a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg) injected intraperitoneally. The pupils were dilated with a mixture of 2.5% Phenylephrine Hydrochloride and 0.5% Tropicamide (Bausch & Lomb, Rochester, NY) ophthalmic solution and a zero-power contact lens (base curvature 3.00 mm, diameter 6.00 mm, optical power 0.00 D; Lakewood, CO 80226) was placed on the eye for imaging. The ORG recordings were performed in the inferonasal region. To minimize motion artifacts caused by respiration and heartbeat, rat’s head was stabilized using a custom-built fixation stage equipped with a bite bar and ear bars.

Data processing
MATLAB (2021a and 2022b, MathWorks, MA, USA) was used for OCT image processing, registration, and phase signal analysis. Python (3.9.2) was used for plotting figures.

The complex-valued OCT signals were reconstructed following the conventional processing pipeline, i.e., k-linearization and discrete Fourier transform of the spectral interferogram captured by the camera. Subpixel-level bulk displacements between the first and subsequent B-scans were estimated by locating the peak of upsampled cross-correlation maps65, then each B-scan was registered to the first B-scan using our custom image registration algorithm61. The registered B-scans were flattened along the IS/OS band. The structural image displayed on a dB scale (Fig. 1A) was obtained by applying 20×log10 to the amplitude component of complex-valued OCT images.

To extract ORG signals, we first calculated the temporal change of phase signals by computing the multiplication of each B-scan with the complex conjugate of the first B-scan,
~ITref(x,z,t) = ~I(x,z,t)~I*(x,z,1) (1)
where ~I(x,z,t) represents the complex-valued OCT images after registration and flattening. x, z, and t denote indices along the lateral, axial, and temporal dimensions, respectively. ~ITref is the time referenced complex-valued OCT signal.

The residual bulk phase errors were canceled out by calculating phase difference between two retinal layers. For each pixel in the target layer, denoted by (xtar, ztar), we selected a reference region from the reference layer—IS/OS in this study. The reference region was centered at xtar and spanned across adjacent 11 A-lines. The temporal phase change in the reference region, φref(xtar, t), can be calculated by,
φref(xtar, t) = ∡ Σz=zb z=zu Σx=xtar+5 x=xtar-5 ~ITref(x,z,t) (2)
where zu and zb denote the upper and lower boundaries of the reference layer, ∡ denotes the argument operator. Note that, to reduce SNR-dependent phase fluctuations when computing phase signals from speckle patterns, we used a two-dimensional reference region instead of a single pixel to calculate φref(xtar, t).

Finally, ORG signals can be obtained by
φtar=ref(xtar, ztar, t) = ∡~ITref(xtar, ztar, t)exp(-jφref(xtar, t)) (3)
where j is the imaginary unit. We then applied the same process to every pixel in the target layer (3 pixels in depth and 512 pixels along the line direction), and calculated the average of extracted phase traces across different pixels, yielding φtar/ref(t). The phase signal can be converted into changes in OPL by,
ΔOPL(t) = λc / 4π φtar=ref(t) (4)
where λc is the center wavelength of OCT imaging beam (840 nm).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "the ΔOPL values shown in Fig. 1E (~180 nm - ~210 nm) appear to\nbe the final ΔOPL values shown in Fig. 1C, rather than the “average of the first 3-4 ms temporal\nwindow.” The latter values would be absolutely smaller, closer to -100 nm.\n[Reply] Figure 1C shows the ΔOPL within the first 3 ms after the stimulus onset. The reported\ncontraction amplitudes were obtained by averaging within the first 3-4 ms temporal window after\nthe stimulus onset, representing the full extent of the rapid contraction. We now clarified it in the\nmanuscripts.\nLine 146: “Amplitudes of the rapid contraction were extracted by averaging ΔOPL at the first 3-\n4 ms temporal window after the stimulus onset…”\n(19) L162-168: Is there a published quantum efficiency for this fast photoreversal? I.e. prior to\nthermodynamic relaxation, what’s the probability of a given photon causing a reversal? I’m not\nsure if that’s a sensible question, and I’m not an expert in this area. Another way to put the\nquestion", "reviewer_2": "Interferometric Imaging of the Reversible Rhodopsin Dynamics in a Living Eye\nZhuo et al.\nReviewer: Daniel X. Hammer\nReview\nZhuo et al. describe a new ORG technique using a high-speed line-scanning OCT system that demonstrates detection of photoisomerization events in the rod-dominated retina of live rats. The authors used short pulses of green light to elicit rapid optical path length (OPL) contraction in rods. They show that the contraction amplitude and latency are relatively constant for stimulus pulses less than 1 ms and remain in the contracted state for hundreds of ms. They also observe that the response is dependent on incident photon density. The authors propose two new models, based on a quasistatic voltage-dependent membrane tension model used for cones, which they update to account for planar and axial viscous damping (planar friction, PF and planar axial friction, PAF models) that is present in densely packed rod discs that are sealed within a tight plasma membrane (and weak or absent in the enfolded cone plasma membrane more amenable to expansion). Finally, the authors show that UV light can partially reverse this contraction and even elicit a contraction on its own. Thus all the rhodopsin isomerization pathways are explored in one study.\nThis wonderful paper is a huge contribution to field of vision research for many reasons. It describes a relatively straightforward technique that can directly probe photoisomerization in live eyes. As a functional tool for vision research and as a disease biomarker, it may prove to exceed the potential of cone-based ORG where the phototransduction contraction response is obscured by the osmotic expansion response. ORG has the potential to replace ERG for live recording of phototransduction signals (akin to the manner in which optogenetics has the potential to replace electrical recordings for brain research), and this paper represents one of the landmark studies to effectuate this on-going technological shift. The paper is extremely clearly written, even for those not directly working in the vision research field. It includes carefully designed and executed experiments. The authors explain very complex photoisomerization models clearly (while also including details of the models' fundamental design in the supplemental section for readers interested in looking under the hood). I highly recommend publication in Nature Communication. The paper can be published as is, and I offer some suggestions below to help clearly present the results and implications of the work.\n1. At some point in the paper, either in the introduction when talking about the elements of the study design, or in the last section of the discussion when talking about clinical translation, it would be helpful if the basic anatomy and physiology of rat retina and differences between rat and human vision could be summarized in a few sentences, e.g., rat vision is rod dominated, UV sensitive cones, etc. at least with respect to the elements that are important for the study.\n2. Speaking of UV sensitive cones, is there any way that this contributes to the signal detected in Fig. 5 that the authors describe in terms of isomerization reversal (or the direct contraction shown in Fig. 5F)? Also, I would say this is the part of the paper that is described in the least detail, and it is perhaps because the authors are least confident about the explanation. For example, I believe the authors are suggesting that the response to direct UV light shown in Fig. 5F is the direct pathway from rhodopsin to Meta II (shown as a dashed line in Fig. 5G but not labeled as such). If this is the hypothesis, it could be described a bit more.\n3. Line 84: Is ultrafast OCT recording necessary? It seems like short pulse duration stimulus pulses are required but given the long stable response of hundreds of ms, the detection speed doesn't need to be that fast. This has implications for clinical translation, where a line-scanning system with its complex optical arrangement may represent a luxury reserved for the vision research domain. Can the authors comment on this as well as other factors on whether this technique can be translated to humans in the periphery where rods density is higher. In rats or humans, is there any crosstalk between cone and rod responses to the stimulation? Will eye motion prohibit similar detection in humans? The current technique doesn't require PR cell visualization. Could it be done in humans with a clinical OCT system outfitted with a stimulus port?\n4. The green flashes produce an exponentially decaying shape (rhodopsin to Meta I) while UV flashes produce an exponentially saturating shape (rhodopsin to Meta II). Is this an additional indication that we are observing two clearly different mechanisms of photoisomerization? Should this be noted somewhere in the paper? Is the shape of the curve indicative on anything?\n5. Fig. 4A indicates the rod disc contracts axially and expands laterally. Obviously, it is easier to detect the axial contraction interferometrically. Is the lateral expansion something that could be detected with AO or is this on a scale that is too small to be seen even with AO? Just curious about this one.\nSmall suggestions and typos:\n1. Lines 43 and 341: UV is listed as 400 nm, which technically isn't UV (maybe 'near-UV'). Also, I believe these lines refer to the citations (other investigator's work), whereas the current paper used 385 nm, which is UV. This is nitpicky but the way the sentences are worded it wasn't immediately clear that the authors were referring to previous papers and not the current work. So the sentences could probably be re-written to be more explicit since the stimulation wavelengths are slightly different.\n2. Lines 129-132: Do the reported contraction amplitudes (averages over time) exclude the initial rapid contraction? That is, was the average taken in the plateau part of the curve? This wasn't clear in the text.\n3. Fig. 6 caption (last line): Correct 'focal'.\n4. Lines 255-257: This sentence is somewhat contradictory, it starts with \"it elicited a completely different ORG response\" and ends with \"similar to the response observed with the green flash\". Suggest rewording so the contradiction isn't so jarring. (Also, later in line 366, the response is described as 'similar to that observed with green stimulus'.)\n5. Line 314: '...a secondary' not '...an secondary'\n6. For clarity, I suggest aligning 1, 2, and 3 in Fig. 5G with the text lines 371-375 (i), (ii), and (iii). Also, 1 and 3 in that figure are the same pathway/mechanism so I am not sure why there should be a distinction between the first and second elongation. Presumably it could be n elongations, correct?\n7. Fig. 6: explicitly show where the OCT red beam enters in the inset (c). Also, does the SLO-CAM have a pinhole not shown (which would be necessary to call it an SLO)?\n8. Supplemental, Line 9: \"...is thermally converted to...\"", "reviewer_3": "This is a strong manuscript and I recommend acceptance for publication after my comments are addressed (see attached).\n\nNature Communications manuscript NCOMMS-25-31811-T\nInterferometric Imaging of the Reversible Rhodopsin Dynamics in a Living Eye\nZhuo et al.\nSummary\nIn this manuscript the authors describe an experiment in which optoretinographic methods (light\nstimuli + dynamic imaging using OCT phase) are employed to demonstrate the reversibility of\nrhodopsin isomerization in the living rat eye. The most significant aspect of the work is the\nreversal of rhodopsin activation, which provides strong evidence that the early outer segment\ncontraction observed in rods and cones is due to rhodopsin activity independent of downstream\nphototransduction steps. It is a very strong result and merits publication in a high-impact journal\nafter addressing a few areas of weakness: 1) the writing is sometimes vague and awkward; 2)\nthere’s no direct reckoning of these results with previous optoretinographic measurements in\nrods; 3) the significance of the finding isn’t properly explained; 4) more OCT characterization\nwould provide better context for the measurements. For the most part, these general criticisms\nare reflected in the specific comments below. However, I am not pointing out every issue with\nwriting mechanics; the authors should read and copyedit carefully before revising.\nComments (indexed by line number)\nTitle: The title should indicate “rat” or “rodent” so as not to imply that this is a clinical result.\nL1-3: This is a very awkwardly phrased description. “[C]onformational changes” in what? This\nis vague. “[T]riggering a phototransduction cascade…” makes it sound like there are several\nphototransduction cascades, and one type of them is triggered by photoismomerazation. Strange\nwording.\nL5+: “Such techniques are invasive, obscured by multiple electrical processes in the retina, and\nin rods, they are limited to the contribution of a small fraction of rhodopsin…” This is\nawkwardly written. I don’t think it’s right to say that the “techniques” are “obscured by multiple\nelectrical processes” or that the “techniques” are limited to the contribution of a small fraction\nof rhodopsin? Also, the first comma should come after the “and,” not before it.\nL18: This should read “A green flash induces a sub-millisecond fast contraction…”\nL26: How does reversible isomerization of photopigment lead to differential diagnosis of\nphotoreceptor dysfunction? There may be a connection, but it would have to be better argued in\nthe main text (see below) to make this connection here.\nL40-41: R1 is the name for the brief depolarizing phase of the ERP. It’s not a “brief depolarizing\nR1 phase.” It should be “a brief depolarizing phase (R1)” or “a brief depolarizing phase termed\n‘R1’” or something like that. Ditto R2.\nL52-54: I’m not conviced that measuring reversibility of rhodopsin isomerization represents a\ndiagnostic tool. Perhaps it does, but it’s not explained here. I would emphasize the basic\nscientific significance and omit the “clinical translatability” or lay out the specific logic of the\nproposed differential diagnosis. This applies to the claim in L96-99 as well.\nL70: One paper missing from the citations list here is Azimipour (2019)1. Azimipour (2020)2\ncould be omitted, since it is derivative from Azimipour (2019)–same system, same methods. The\n2019 paper is more appropriate to cite because it’s in a higher impact journal (Optica) and it\npredates Zhang (2019) and Azimipour (2020). An alternative would be to simply cite Hillmann\n(2016)3, which lays out the key principles and from which all the other work is derived. Or\nperhaps Hillmann (2016) and a review, since the number of ORG papers is growing rapidly.\nL56-63: This is a long and confusing sentence.\nL63-65: The “overshadowed” is confusing here. The only technique mentioned thus far is patch-\nclamp recordings, in which no class of cells should overshadow another. Could you mean in\nthe ERG recording now? If so, it should be stated more clearly. This applies to line 52+ above too–\npresumably recordings in DR and RP must be ERG and not patch-clamp recordings.\nL66-70: It would be appropriate to cite some antecedant work here including spectral-domain\nphase microscopy4, self-referenced phase imaging5, and perhaps even phase-based bulk-motion\nestimation6.\nL77-79: Most ORG papers state putative osmotic causes for elongation, but a recent paper,\nTomczewski (2025)7, suggests that molecular mechanical forces may play a role too. This should\nprobably be mentioned as well, esp. given the role of inter-disc spacers in the present\nmanuscript’s theory of contraction. That paper is cited earlier, but should be mentioned here in\nthe summary of ORG hypotheses.\nL96-99: See L52-54 above.\nL118-119: Please provide a citation or explanation justifying segmentation of ROST from RPE\nby using the “top of the thick hyperreflective band.” In histological images, the top of that band\nlooks like a mess of ROST, RPE apical processes, RPE apical surface, phagosomes, and other\nstuff. Also, it would be good to see a diagram illustrating how much of the thick hyperreflective\nband was used. This is hinted at in Fig. 1A, but it would be good to see a clear picture (either B-\nscan or axial reflectance profile, i.e., B-scan averaged laterally) of the portion of the thick\nhyperreflective band that was used for ΔOPL measurements.\nL118-119: Did the authors use any of the ML classification used by Tan (2024)8? According to\nthat paper, while there was a tendency for pixels exhibiting ROS behavior to lie near the top of\nthe hyperreflective band, the depth distributions of the various signal types were broad and\noverlapped significantly. The the authors of the present manuscript notice any of the pixels\nselected for analysis “misbehaving,” i.e., acting like RPE pixels instead of ROST pixels?\nL118-119: The authors should state the SNR range of the IS/OS and ROST pixels. This\ndetermines the theoretical phase sensitivity. This is related to the “L402+ and Fig. 1A” comment\nbelow.\nL129-130 and Fig. 1C", "reviewer_perhaps": "why can the photoreversal be neglected for long stimuli, but not short ones,\nand how do you calculate the lower exposure time limit for negligible photoreversals?\n[Reply] The absorption coefficients of relevant rhodopsin isoforms and the quantum efficiencies\nof photoreversal are summarized in Table S1. For example, the quantum efficiency of the transition\nfrom Meta I back to rhodopsin is 0.33, about half that of rhodopsin activation (0.67). These fast\nphotoreversal processes apply only to early photoproducts up to Meta I, which converts to Meta II\nwithin a few hundreds of microseconds. Therefore, the fast photoreversal effect is pronounced\nonly when a large number of photons are delivered while a substantial portion of rhodopsin\nisoforms have not yet decayed to Meta II –i.e., under conditions of high-intensity, short-duration\nstimulation. We have revised the manuscript to improve clarity.\nLine 179: “Early photoproducts, up to Meta I, can absorb a second photon and restore either\nrhodopsin or isorhodopsin, a process called photoreversal [36-38]. Therefore, if a large\nnumber of photons are delivered while a substantial portion of rhodopsin isoforms\nhave not yet decayed to Meta II, photoreversal of the rhodopsin bleaching cannot be\nneglected. Given that the flash durations in our experiments are comparable to the\nthermal decay time constant from Meta I to Meta II (a few hundred microseconds in\nrodents [5, 9]), we considered all critical light-driven and thermal processes involved\nin flash photolysis to determine the bleach levels (Fig. 3A and Supplementary\nInformation: Bleach level calculation).”\n(20) L200: It’s incorrect to call the cytoplasm “viscous” in general. Its viscosity is low compared to\nmany other liquids, and similar to that of water.\n[Reply] We have removed the word “viscous” before “cytoplasm” in both the revised manuscript\nand the supplementary information.\n(21) L202: “... drive axially the discs and the ROST” is very confusing.\n[Reply] For clarify, we have revised it to “causes discs and ROST to move axially”.\n(22) L300+: The authors should speculate why the light-evoked deformations reported here differ\nfrom previous reports on human rods. Pfaffle (2019)9 shows a B-scan in which a distinct ROST\nlayer is visible, and the ΔOPL reported there is monotonic and positive. Of course their\nbandwidth (~100 Hz) is much lower than the present manuscript, but this paper’s Fig. 5A shows\nthat the ROS contraction lasts at least 200 ms, and suggests that it lasts much longer–long\nenough to be observed at 120 Hz. The volume rate of Azimipour (2020), 6 Hz, is even slower,\nbut they also reported significant elongation, with the benefit of spatial resolution of rods. Even\nat 6 Hz they should observe a contraction. Both of those findings were confirmed by model-\nbased classification of arbitrary OS signals in Pfaffle (2024)10. Segmentation of the rod layer and\nisolation of the ROS response seems more tenuous in the present work than in any of those\npapers. The authors should comment on these apparent inconsistencies. I don’t mean to cast\ndoubt on the present results; I find them more compelling than the earlier rod measurements,\nesp. in light of the photoreversal effects. Nevertheless, the inconsistency should be addressed.\n[Azimipour (2020)] Azimipour M, Valente D, Vienola KV, et al. Optoretinogram: Optical Measurement of Human Cone and\nRod Photoreceptor Responses to Light. Optics Letters. 2020;45(17):4658- 4661. doi:10.1364/OL.398868.\n9. Pfäffle C, Spahr H, et al. Simultaneous Functional Imaging of Neuronal and Photoreceptor Layers in Living Human Retina.\nOptics Letters. 2019;44(23):5671-5674. doi:10.1364/OL.44.005671.\n10. Pfäffle C, Puyo L, Spahr H, Hillmann D, Miura Y, Hüttmann G. Unraveling the functional signals of rods and cones in the\nhuman retina: Separation and analysis. Frontiers in Ophthalmology. 2024;4. doi:10.3389/fopht.2024.1340692.\n[Reply] A recent study in human subjects employed AO line-scan OCT to identify rod photoreceptors\nat 10° eccentricity and successfully measured the rapid contraction in human rod OS [Li et al., 2025].\nNotably, the duration of this rapid contraction (~100 ms at 3.3% bleach level and ~300 ms for at 39.4%\nbleach level) is much shorter than those observed in rodents rod OS. Prior studies on human rod ORG\nwere conducted with relatively low temporal resolution – 6 Hz in Azimipour (2020) and 8 Hz in Pfäffle\n(2019, 2024). The limited temporal resolution, along with relatively low bleach levels (≤1% in\nPfäffle (2024) and ≤4% in Azimipour (2020)) or seconds-long low power visual stimulation\n[Pfäffle, 2019], likely explain the absence of rapid contraction signals in those studies. We have\nadded these considerations to Discussion part D.\n[Li et al., 2025] H. Li, C. E. Weiss, V. P. Pandiyan, D. Nanni, T. Liu, P. W. Kung, B. Tan, V. A. Barathi, L. Schmetterer, R.\nSabesan, and T. Ling, “Optoretinography reveals rapid rod photoreceptor movement upon photoisomerization,” bioRxiv, p.\n2025.03.22.644466, 2025.\n(23) L410+: Please describe the power incident on the retina and the length of the irradiated line\non the retina. Combined with the 512 pixel spatial dimension on the camera, this would tell us the\nsampling density. It would be good to state the diffraction-limited spot size on the retina as well,\nsince the relationship between spatial resolution and sampling is an important operational\nparameter.\n[Reply] The incident power on the cornea was 2.8 mW, and the irradiated line length on the retina\nwas estimated to be 330 µm. The diffraction-limited resolution (FWHM) was estimated to be 3.6\nµm. These details have been added to the revised manuscript.\nLine 493: “The irradiated line length on the retina was estimated to be 330 µm, and the\ndiffraction-limited resolution (FWHM) was estimated to be 3.6 µm.”\n(24) L506+: It is not clear whether the same 2D reference region is used as reference phase for all\nthe target pixels, or whether each target pixel is analyzed with respect to its own 2D reference\nregion. Also, please state rationale for using a 2D reference region instead of reference pixels\nwithin the target pixel’s A-line (which, I believe, is the standard phase-based ORG method,\ne.g. Pandiyan (2020)11 or Zhang (2019)12.\n11. Pandiyan VP, Bertelli AM, Kuchenbecker J, et al. The Optoretinogram Reveals How Human Photoreceptors Deform in\nResponse to Light. bioRxiv. 2020:2020.01.18.911339. doi:10.1101/2020.01.18.911339.\n12. Zhang F, Kurokawa K, Lassoued A, Crowell JA, Miller DT. Cone Photoreceptor Classification in the Living Human Eye\nfrom Photostimulation-Induced Phase Dynamics. Proceedings of the National Academy of Sciences. 2019;116(16):7951-\n7956.\n[Reply] Each pixel was analyzed relative to its own 2D reference region. We found that the symbol\non the left-hand side of the original Eq. 2, 𝜑 (𝑡), may cause confusion, as it should have been\nref\nwritten as 𝜑 (𝑥 ,𝑡) to reflect its dependence on lateral position. This have been updated in Eqs.\nref tar\n2 and 3 in the revised manuscript.\nA 2D reference region was used to reduce SNR-dependent phase fluctuations when extracting\nphase signals from speckle patterns. Such strategy was also employed in a previous ORG study\n[Tan et al., 2024]. We have clarified it in the revised manuscript.\n[Tan et al., 2024] Tan B, Li H, Zhuo Y, et al. Light-evoked deformations in rod photoreceptors, pigment epithelium and\nsubretinal space revealed by prolonged and multilayered optoretinography. Nature Communications. 2024;15(1):5156.\nLine 586: “Note that, to reduce SNR-dependent phase fluctuations when computing phase signals\nfrom speckle patterns, we used a two-dimensional reference region instead of a single\npixel to calculate 𝜑 (𝑥 ,𝑡).”\nref tar\n(25) L509 and Eq. 2: should be “upper” and “lower,” or “top” and “bottom,” but not “upper” and\n“bottom”.\n[Reply] We have revised it to “the upper and lower boundaries of the reference layer”.\n(26) L514-515: Please state how many traces are averaged. This number, in conjunction with the\nSNR of the IS/OS and ROST layers, is important for making sense of the phase noise (i.e., the gray\nfilled-in regions in the plots).\n[Reply] The target layer consists of 3 pixels in depth and 512 pixels along the line direction. This\ninformation has been added to the revised manuscript. We would like to clarify that the gray filled-\nin regions (standard deviations) in the plots were also affected by inter-individual difference, as\nsignals were measured from different rats.\nLine 591: “We then applied the same process to every pixel in the target layer (3 pixels in depth\nand 512 pixels along the line direction), and calculated the average of extracted phase\ntraces across different pixels ...”\nWe would like to thank the reviewers for carefully reading our revisions. Please 8ind below our\nresponses (blue) and corresponding revisions in the manuscript (red).\nReviewer #1:\n(1) The authors have addressed all my comments satisfactorily.\nHowever, I propose to change the response to comment (17) of reviewer 3:\nThe logarithmic scaling of the OCT signal often leads to confusions as mentioned by the reviewer.\nThe sentence “Intensity (dB) was calculated as 10×log10 of the OCT signal intensity” may as well\nbe misleading since \"OCT signal intensity\" in not clearly defined. The accepted standard is, as used\nhere, 20 Log10 of the absolute value of the Fourier transform the interference spectrum. Hence,\nthe added comment may even be confusing. I would suggest to add to Section IV D the explanation,\nthat OCT signal was calculated by 20 Log10 of the absolute value of the Fourier transform instead\nof changing the caption of Fig. 1.\n[Reply] We thank the reviewer for the suggestion. In the revised manuscript, we have removed the\nwording “Intensity (dB) was calculated as 10×log10 of the OCT signal intensity” from the caption\nof Fig. 1 and clarified the definition of the dB-scale OCT image in the Methods section.\nLine 564: “The structural image displayed on a dB scale (Fig. 1A) was obtained by applying\n20×log10 to the amplitude component of complex-valued OCT images.”", "reviewer_author_response": "We would like to thank the reviewers for carefully reading our manuscript and providing\ndetailed comments. Please :ind below our point-by-point responses (blue) and corresponding\nrevisions in the manuscript (red).\nReviewer #1:\n(1) Although rat retina is dominated by rods the contribution of cone should be discussed more\nquantitatively. How strong is the confounding effect? How can contributions of cones and rods be\nseparated. This is especially important for human applications.\n[Reply] In rats, ~99% of photoreceptors are rods [Szél et al., 1992], so cones are unlikely to have\na noticeable impact on the measured ORG signals. We have added a sentence in the Introduction\nto clarify this point.\n[Szél et al., 1992] Á. Szél and P. Röhlich, “Two cone types of rat retina detected by anti-visual pigment antibodies,”\nExperimental Eye Research, vol. 55, no. 1, pp. 47–52, 1992\nLine 94: “In this study, we investigated ORG signatures associated with rhodopsin dynamics in\nthe living rat eye, where ~99% of photoreceptors are rods [Szél et al., 1992], making it\nan ideal model for studying rod ORG signals.”\nPotential strategies to differentiate rod responses from cone responses in human applications are\ndiscussed in our reply to comment (4) below.\n(2) In my opinion, the description of the results and the discussion relies too much on the\nsupplement. E.g. comparison of the fitted parameter with literature (lines 189 - 192), comparison\nof model with and without viscous damping (lines 218 - 222).\n[Reply] We have added more details about the voltage-dependent membrane tension model,\nelaborated on the fitted parameters of the quasistatic model, and revised the text so that the\ncomparison between the models with and without viscous damping is based on Figs. 4B and 4C in\nthe manuscript.\nLine 204: “The cumulative contraction of approximately a thousand discs results in the\ncontraction of the photoreceptor OS by tens or even hundreds of nanometers.”\nLine 209: “At the best fit, we obtained an initial membrane tension 𝜏 of 0.59 µN∙m-1 and a\n!\nbending modulus 𝜅 =0.23×10%&' N∙m – within the ranges of the membrane surface\n$\ntension and bending modulus in the literature: 0.1–1 µN∙m-1 and 0.5−2×10%&' N∙m\n[30], respectively.”\nLine 240: “As shown in Figs. 4D and 4E, the viscoelastic model accurately predicts the latency of\nthe maximum contraction for various pulse durations and energy levels, whereas the\nquasistatic model exhibits much faster dynamics (blue curves in Figs. 4B and 4C).”\n(3) Four different schemes (two in the manuscript and two in the supplement) are used to describe\nthe role and transitions of the different rhodopsin states. A unification would be very helpful. Fig.\n3 (A) in the manuscript does not help for the understanding, since the mathematical model is only\ndescribed in the supplement.\n[Reply] We have combined the original Fig. 5G and two supplementary figures into a new Fig. 6 in\nthe revised manuscript, and the relevant text has been updated accordingly. We also kept a\ndiagram in Fig. 3A to help readers gain an intuitive understanding of the photoreversal process.\n(4) In the discussion, part D, the problem of discrimination of rod and cone distribution in humans\nshould be addressed when discussing translation.\n[Reply] We have added a paragraph in Discussion part D to elaborate on anatomical differences\nbetween rat and human retinas, as well as potential strategies for extracting rod ORG responses in\nhuman applications.\nMinor remarks:\n(1) For stimulation strength irradiance instead of radiant exposure should be stated. In Fig. 5\nvalues with different number of valid digits (340 µJ and 336 µJ) are given.\n[Reply] We have added the stimulation strength irradiance and corrected the value in the caption\nof Fig. 5A from 336 µJ to 340 µJ.\nLine 123: “Considering a transmittance of 0.77 through the anterior segment of the eye at 520\nnm [33,34] and an illumination area on the retina of 1.88 mm2, this corresponds to\napproximately 5.4×10( photons⁄µm2 incident on the retina.”\nCaption of Fig. 2:\n“ROS contraction with varying pulse energies (2.40 – 77.50 µJ, corresponding to photon\ndensities of 2.6×10* – 8.3×10( photons⁄µm2 on the retina).”\nLine 254: “Pulse energy of the green flash was sufficiently high (340 µJ, 3.6×10+ photons⁄μm2\non the retina) to bleach nearly all rhodopsin, ...”\nLine 260: “UV flashes were delivered 100 ms after each green pulse at UV energy levels of 30, 70,\n112 and 224 µJ, measured in front of the cornea. As shown in Fig. 5B, the first UV flash\n(1 ms, 112 µJ, 7.5×10( photons⁄μm2 on the retina), applied to an extensively\nbleached retina, elicited a pronounced elongation of the ROS (with ~10 ms time\nconstant)”\nCaption of Fig. 5:\n“(A) Control experiment: ORG responses elicited by two green flashes (1 ms, 340 µJ\nmeasured in front of the cornea, corresponding to 3.6×10+ photons⁄µm2 on the\nretina) delivered 200 ms apart. (B) Response to a flash sequence of green-UV-green-\nUV, with a UV flash of 1 ms, 112 µJ. Given a transmittance of 0.54 through the anterior\nsegment of the eye at 385 nm [33,34] and an illumination area on the retina of 1.56\nmm2, this corresponds to approximately 7.5×10( photons⁄µm2 incident on the\nretina.”\n(2) Line 189: Cite Fig. 3(B) instead of S1.\n[Reply] We would like to clarify that the meanings of the gray curves in Fig. S1 and Fig. 3B are\ndifferent: the curve in Fig. S1 represents the prediction from the voltage-dependent membrane\ntension model, whereas the curve in Fig. 3B is simply a logarithmic fit. Since our aim here is to\ncompare the experimental results and the model prediction, we refer to Fig. S1.\n(3) Line 360: Transition from Meta I to Meta II is by deprotonation.\n[Reply] We have corrected this error in the revised manuscript.\n(4) Line 419: The Badal platform should be explained for scientist not working in the field.\n[Reply] The lens L5 and the animal fixation stage were mounted on a Badal platform, such that\ntranslating the platform alters the vergence of the beam entering the eye, enabling compensation\nfor subject-induced defocus. This explanation has been added to the revised manuscript.\nLine 488: “The lens L5 and the animal fixation stage were mounted on a Badal platform, such that\ntranslating the platform alters the vergence of the beam entering the eye, enabling\ncompensation for subject-induced defocus.”\n(5) Line 438: Give type and manufacturer of the SLO camera.\n[Reply] The camera was spL4096-140km from Basler. This information has been added to the\nrevised manuscript.\nLine 511: “The line-scan ophthalmoscope (LSO) camera (spL4096-140km, Basler) was aligned\nto be parfocal with the OCT camera.”\n(6) Line 484: At which site of the retina were the measurements done.\n[Reply] The measurement was performed in the inferonasal region. This has been clarified in the\nrevised manuscript.\nLine 555: “The ORG recordings were performed in the inferonasal region.”\nReviewer #2:\n(1) At some point in the paper, either in the introduction when talking about the elements of the\nstudy design, or in the last section of the discussion when talking about clinical translation, it would\nbe helpful if the basic anatomy and physiology of rat retina and differences between rat and human\nvision could be summarized in a few sentences, e.g., rat vision is rod dominated, UV sensitive cones,\netc. at least with respect to the elements that are important for the study.\n[Reply] We have added a paragraph in Discussion part D to clarify the anatomical differences\nbetween rat and human retinas.\n(2) Speaking of UV sensitive cones, is there any way that this contributes to the signal detected in\nFig. 5 that the authors describe in terms of isomerization reversal (or the direct contraction shown\nin Fig. 5F)? Also, I would say this is the part of the paper that is described in the least detail, and it\nis perhaps because the authors are least confident about the explanation. For example, I believe\nthe authors are suggesting that the response to direct UV light shown in Fig. 5F is the direct\npathway from rhodopsin to Meta II (shown as a dashed line in Fig. 5G but not labeled as such). If\nthis is the hypothesis, it could be described a bit more.\n[Reply] Given that S-cones constitute only a small fraction (~0.05%) of photoreceptors in rats [Szél\net al., 1992], and that rhodopsin absorption at 385 nm is still approximately 20% of its peak\nabsorption in the green range [Sakai et al., 2022], interference from S-cones should be minimal.\nThese pieces of information have been added to the revised manuscript.\n[Szél et al., 1992] Á. Szél and P. Röhlich, “Two cone types of rat retina detected by anti-visual pigment antibodies,”\nExperimental Eye Research, vol. 55, no. 1, pp. 47–52, 1992.\n[Sakai et al., 2022] K. Sakai, Y. Shichida, Y. Imamoto, and T. Yamashita, “Creation of photocyclic vertebrate rhodopsin by\nsingle amino acid substitution,” eLife, vol. 11, p. e75979, 2022.\nLine 398: “Given that S-cones constitute only a small fraction (~0.05%) of photoreceptors in rats\n[Szél et al., 1992], and that rhodopsin absorption at 385 nm is still approximately 20%\nof its peak absorption in the green range [Sakai et al., 2022], interference from S-cones\nshould be minimal. Therefore, the rapid contraction in Fig. 5F is more likely to originate\nfrom the transition from rhodopsin to Meta II.”\n(3) Line 84: Is ultrafast OCT recording necessary? It seems like short pulse duration stimulus\npulses are required but given the long stable response of hundreds of ms, the detection speed\ndoesn't need to be that fast. This has implications for clinical translation, where a line-scanning\nsystem with its complex optical arrangement may represent a luxury reserved for the vision\nresearch domain. Can the authors comment on this as well as other factors on whether this\ntechnique can be translated to humans in the periphery where rods density is higher. In rats or\nhumans, is there any crosstalk between cone and rod responses to the stimulation? Will eye motion\nprohibit similar detection in humans? The current technique doesn't require PR cell visualization.\nCould it be done in humans with a clinical OCT system outfitted with a stimulus port?\n[Reply] Ultrafast recording is critical for capturing the full temporal dynamics/transients of\nunknown signals in scientific investigations aimed at uncovering underlying mechanisms, as well\nas in preclinical studies for identifying potential disease biomarkers. However, such high temporal\nresolution may be less essential for clinical applications.\nOne challenge in translating this study to human subjects is to differentiate rod ORG signals from\ncone ORG signals. Recently, a study in human subjects employed AO line-scan OCT to identify rod\nphotoreceptors at 10° eccentricity and successfully measured rapid contraction in human rod OS\n[Li et al., 2025, bioRxiv]. Notably, the duration of this rapid contraction (~100 ms at 3.3% bleach\nlevel and ~300 ms at 39.4% bleach level) is much shorter than that observed in rodent rod OS. One\npossible explanation is that the elongation of surrounding cones may affect the rod OS responses.\nIn future studies, it would be interesting to measure human rod OS responses in peripheral retina,\nwhere cones density is much lower [Curcio et al., 1990], to further investigate potential crosstalk\nbetween rods and cones. Additionally, as suggested by the reviewer, resolving photoreceptors may\nnot be necessary in the peripheral retina due to the high proportion of rods, which could allow\nnon-AO OCT systems to detect the rapid OS contraction in human rods. Another major challenge\nin conducting ORG experiments using clinical OCT systems is the signal decorrelation caused by\nhead and eye movements. Several strategies–including improved post-processing algorithms\n[Spahr et al., 2019; Vienola et al., 2022; Li et al., 2025, J. Phys. D Appl. Phys.], active eye-tracking\n[Wong et al., 2025], and new OCT recording protocols [Gong et al., 2024]–are being explored to\nmitigate signal decorrelation during ORG measurements and facilitate the clinical translation of\nORG techniques.\nWe have added a paragraph in Discussion part D to elaborate on these points.\n[Li et al., 2025] H. Li, C. E. Weiss, V. P. Pandiyan, D. Nanni, T. Liu, P. W. Kung, B. Tan, V. A. Barathi, L. Schmetterer, R.\nSabesan, and T. Ling, “Optoretinography reveals rapid rod photoreceptor movement upon photoisomerization,” bioRxiv, p.\n2025.03.22.644466, 2025.\n[Curcio et al., 1990] C. A. Curcio, K. R. Sloan, R. E. Kalina, and A. E. Hendrickson, “Human photoreceptor topography,” Journal\nof Comparative Neurology, vol. 292, no. 4, pp. 497–523, 1990.\n[Spahr et al., 2019] H. Spahr, C. Pfaffle, S. Burhan, L. Kutzner, F. Hilge, G. Huttmann, and D. Hillmann, “Phase-sensitive\ninterferometry of decorrelated speckle patterns,” Sci Rep, vol. 9, no. 1, p. 11748, 2019.\n[Vienola et al., 2022] K. V. Vienola, D. Valente, R. J. Zawadzki, and R. S. Jonnal, “Velocity-based optoretinography for clinical\napplications,” Optica, vol. 9, no. 10, pp. 1100–1108, 2022.\n[Li et al., 2025] H. Li, B. Tan, V. P. Pandiyan, V. A. Barathi, R. Sabesan, L. Schmetterer, and T. Ling, “Phase-restoring subpixel\nimage registration: enhancing motion detection performance in fourier-domain optical coherence tomography,” J. Phys. D Appl.\nPhys., vol. 58, no. 14, p. 145102, Apr. 2025.\n[Wong et al., 2025] J. H. Wong, S. Luo, Z. Hosseinaee, F. Feroldi, and A. Roorda, “Optoretinography with actively stabilized\nadaptive optics optical coherence tomography,” Biomedical Optics Express, vol. 16, no. 8, pp. 3222–3236, 2025.\n[Gong et al., 2024] Z. Gong, Y. Shi, J. Liu, R. Sabesan, and R. K. Wang, “Light-adapted flicker-optoretinography based on raster-\nscan optical coherence tomography towards clinical translation,” Biomedical Optics Express, vol. 15, no. 10, pp. 6036–6051, 2024.\n(4) The green flashes produce an exponentially decaying shape (rhodopsin to Meta I) while UV\nflashes produce an exponentially saturating shape (rhodopsin to Meta II). Is this an additional\nindication that we are observing two clearly different mechanisms of photoisomerization? Should\nthis be noted somewhere in the paper? Is the shape of the curve indicative on anything?\n[Reply] We would like to clarify that the rapid contraction was induced by the transition from\nrhodopsin to Meta II, while the rapid elongation was caused by the transition from Meta II to Meta\nIII. Amplitude of both responses saturates with the stimulus strength – just one is negative\n(contraction), while ethe other is positive (elongation). We have added a sentence to the\nmanuscript to highlight these two different rhodopsin isomerization pathways.\nLine 248: “While the hyperpolarizing phase of the ERP is associated with the transition from\nMeta I to Meta II, near-UV photons can induce a reversed conformational change from\nMeta II to Meta III [10].”\n(5) Fig. 4A indicates the rod disc contracts axially and expands laterally. Obviously, it is easier to\ndetect the axial contraction interferometrically. Is the lateral expansion something that could be\ndetected with AO or is this on a scale that is too small to be seen even with AO? Just curious about\nthis one.\n[Reply] Considering a rod outer segment (ROS) with a diameter of 1.7 µm and a length of 24 µm, a\n100 nm axial contraction corresponds to a 3.5 nm increase in its diameter. While AO allows the\nvisualization of individual photoreceptors, directly measuring such a small change in ROS diameter\nremains challenging.\nSmall suggestions and typos:\n(1) Lines 43 and 341: UV is listed as 400 nm, which technically isn't UV (maybe 'near-UV'). Also, I\nbelieve these lines refer to the citations (other investigator's work), whereas the current paper\nused 385 nm, which is UV. This is nitpicky but the way the sentences are worded it wasn't\nimmediately clear that the authors were referring to previous papers and not the current work. So\nthe sentences could probably be re-written to be more explicit since the stimulation wavelengths\nare slightly different.\n[Reply] We have revised the relevant content for better clarify.\nLine 43: “Previous electrical recordings in living rat eyes demonstrated that a 400-nm flash\ndelivered to photoreceptors fully bleached by green light can elicit a rapid electrical\nsignal of opposite polarity, indicating that near-UV photons can revert the activated\nrhodopsin into another isoform [9], ...”\nLine 246: “Previous studies have demonstrated that a near-UV flash delivered to Meta II can elicit\nan electrical signal of opposite polarity compared to typical ERP [9, 45].”\nLine 370: “Previous electrical recordings demonstrated that an intense 400-nm stimulus applied\nto a fully bleached retina induces electrical response opposite in polarity to the typical\nERP [9, 45]. Moreover, a second green flash following the 400-nm pulse elicits again an\nERP response with the same shape as that of the pre-bleached retina (albeit with a\nlower amplitude), suggesting that photopigment was regenerated by the near-UV flash.\nThe diagram of the photoisomerization and thermal relaxation pathways in rhodopsin\nshown in Fig. 6 suggests that (near-)UV light can trigger 15-anti/15-syn isomerization\nin Meta II ...”\n(2) Lines 129-132: Do the reported contraction amplitudes (averages over time) exclude the initial\nrapid contraction? That is, was the average taken in the plateau part of the curve? This wasn't clear\nin the text.\n[Reply] The reported contraction amplitudes certainly include the full extent of the rapid\ncontraction – they were measured between 3 and 4 ms after the stimulus, when rapid contraction\nphase reaches its plateau, as illustrated in Fig. 1C. We now clarified it in the manuscripts.\nLine 146: “Amplitudes of the rapid contraction were extracted by averaging ΔOPL at the first 3-\n4 ms temporal window after the stimulus onset…”\n(3) Fig. 6 caption (last line): Correct 'focal'.\n[Reply] We have corrected this typo.\n(4) Lines 255-257: This sentence is somewhat contradictory, it starts with \"it elicited a completely\ndifferent ORG response\" and ends with \"similar to the response observed with the green flash\".\nSuggest rewording so the contradiction isn't so jarring. (Also, later in line 366, the response is\ndescribed as 'similar to that observed with green stimulus'.)\n[Reply] We have revised the sentence to for better clarify.\nLine 282: “Remarkably, the UV flash evoked different ORG responses in light-adapted (Fig. 5B)\nand dark-adapted retinas (Fig. 5F). When delivered to a dark-adapted retina without a\npreceding green pulse, the UV flash elicited a rapid contraction by 200 nm (Fig. 5F),\nsimilar to the response observed with a green flash.”\n(5) Line 314: '...a secondary' not '...an secondary'\n[Reply] We have corrected this typo.\n(6) For clarity, I suggest aligning 1, 2, and 3 in Fig. 5G with the text lines 371-375 (i), (ii), and (iii).\nAlso, 1 and 3 in that figure are the same pathway/mechanism so I am not sure why there should\nbe a distinction between the first and second elongation. Presumably it could be n elongations,\ncorrect?\n[Reply] As suggested by the Reviewer #1, Fig. 5G has been replaced with a new Fig. 6 showing the\nrhodopsin isomerization pathways under green and UV flashes. Note that processes ①–③ in Figs.\n5B-E are distinct from processes (i) – (iii) described in lines 371-375 (current lines 406-408).\nSpecifically, ①–③ in Figs. 5B-E refer to the dynamics evoked by a sequence of green-UV-green-\nUV flashes, where the mechanisms underlying the first and second elongation are the same.\nHowever, points (i) – (iii) describe three simultaneous processes induced by a second UV flash\nafter a preceding UV flash (Fig. 5F).\n(7) Fig. 6: explicitly show where the OCT red beam enters in the inset (c). Also, does the SLO-CAM\nhave a pinhole not shown (which would be necessary to call it an SLO)?\n[Reply] We have added the OCT red beam to the inset (C). The term “scanning laser\nophthalmoscope (SLO)” used in the original manuscript should have been “line-scan\nophthalmoscope (LSO)”, and this has been corrected in both the main text and Fig. 7. The slit shown\nin Fig. 7A was used to suppress back reflections from the lenses and to provide partial confocality.\nLine 511: “The line-scan ophthalmoscope (LSO) camera (spL4096-140km, Basler) was aligned\nto be parfocal with the OCT camera. LSO imaging was used for finding a focal plane of\nthe retina, while adjusting the Badal platform, prior to OCT imaging.”\n(8) Supplemental, Line 9: \"...is thermally converted to...\"\n[Reply] We have corrected this grammatical error.\nReviewer #3:\n(1) Title: The title should indicate “rat” or “rodent” so as not to imply that this is a clinical result.\n[Reply] We have revised the title to “Interferometric Imaging of the Reversible Rhodopsin\nDynamics in the Living Rat Eye” to better reflect the scope of this study.\n(2) L1-3: This is a very awkwardly phrased description. “[C]onformational changes” in what? This\nis vague. “[T]riggering a phototransduction cascade...” makes it sound like there are several\nphototransduction cascades, and one type of them is triggered by photoismomerazation. Strange\nwording.\n[Reply] To improve clarify, we have rephrased the sentence as “Vision begins with\nphotoisomerization and further conformational changes of photopigments, followed by the\nphototransduction cascade in photoreceptors”.\n(3) L5+: “Such techniques are invasive, obscured by multiple electrical processes in the retina, and\nin rods, they are limited to the contribution of a small fraction of rhodopsin...” This is\nawkwardly written. I don’t think it’s right to say that the “techniques” are “obscured by multiple\nelectrical processes” or that the “techniques” are limited to the contribution of a small fraction\nof rhodopsin? Also, the first comma should come after the “and,” not before it.\n[Reply] We have revised the sentence to “Microelectrode recordings revealed an early receptor\npotential (ERP) accompanying these conformational transitions. In rods, the measured electrical\nsignals are limited to the contribution of a small fraction of rhodopsin embedded in the nascent\ndisc membrane and plasma membrane.”\n(4) L18: This should read “A green flash induces a sub-millisecond fast contraction...”\n[Reply] We have revised it to “A green flash induces a rapid (sub-millisecond) contraction of the\nouter segments by hundreds of nanometers ...”\n(5) L26: How does reversible isomerization of photopigment lead to differential diagnosis of\nphotoreceptor dysfunction? There may be a connection, but it would have to be better argued in\nthe main text (see below) to make this connection here.\n[Reply] We added a clarification in the Discussion (see below) and changed the wording here as\nfollowing: “to more specific clinical diagnosis”.\n(6) L52-54: I’m not convicted that measuring reversibility of rhodopsin isomerization represents\na diagnostic tool. Perhaps it does, but it’s not explained here. I would emphasize the basic scientific\nsignificance and omit the “clinical translatability” or lay out the specific logic of the proposed\ndifferential diagnosis. This applies to the claim in L96-99 as well.\n[Reply] We added a clarification in the Discussion (see below) and changed the words “differential\ndiagnosis” here with: “more specific clinical diagnosis”.\n(7) L96-99: See L52-54 above.\n[Reply] We now added a clarification to the section “ORG vs. ERG: translation to clinic” in\nDiscussion.\nLine 434: “Different signatures of the isomerization (ERP, contraction of the OS) and\nphototransduction (LRP, expansion of OS in cones and under low stimuli in rods)\nenable differential diagnostics of the various aspects of photoreceptor’s dysfunction.”\n(8) L40-41: R1 is the name for the brief depolarizing phase of the ERP. It’s not a “brief depolarizing\nR1 phase.” It should be “a brief depolarizing phase (R1)” or “a brief depolarizing phase termed ‘R1’”\nor something like that. Ditto R2.\n[Reply] We revised the sentence to “...: a brief depolarizing phase (R1) corresponding to the\nformation of Meta I, and a dominant hyperpolarizing phase (R2) reflecting the conversion from\nMeta I to Meta II”. We have also corrected relevant expressions in other parts of the manuscript\nand supplementary information.\n(9) L70: One paper missing from the citations list here is Azimipour (2019)1. Azimipour (2020)2\ncould be omitted, since it is derivative from Azimipour (2019)–same system, same methods. The\n2019 paper is more appropriate to cite because it’s in a higher impact journal (Optica) and it\npredates Zhang (2019) and Azimipour (2020). An alternative would be to simply cite Hillmann\n(2016)3, which lays out the key principles and from which all the other work is derived. Or perhaps\nHillmann (2016) and a review, since the number of ORG papers is growing rapidly.\n1. Azimipour M, Migacz JV, Zawadzki RJ, Werner JS, Jonnal RS. Functional Retinal Imaging Using Adaptive Optics Swept-\nSource OCT at 1.6 MHz. Optica. 2019;6(3):300-303.\n2. Azimipour M, Valente D, Vienola KV, et al. Optoretinogram: Optical Measurement of Human Cone and Rod Photoreceptor\nResponses to Light. Optics Letters. 2020;45(17):4658- 4661. doi:10.1364/OL.398868.\n3. Hillmann D, Spahr H, Pfäffle C, Sudkamp H, Franke G, Hüttmann G. In Vivo Optical Imaging of Physiological Responses to\nPhotostimulation in Human Photoreceptors. Proceedings of the National Academy of Sciences of the United States of America.\n2016;113(46):13138- 13143. doi:10.1073/pnas.1606428113.\n[Reply] The 2020 BOE paper by Azimipour et al. was included because it reported human rod ORG\nsignals, which have so far been investigated in only a few papers. We have also added the 2019\nOptica paper to the list of citations.\n(10) L56-63: This is a long and confusing sentence.\n[Reply] We have rephrased the sentence as “Even though rods constitute the vast majority of\nphotoreceptors in most mammalian retinas [12], such electrical recordings from rods are more\nchallenging than from cones due to the distinct membranous organization of their outer segments.\nSpecifically, most of rhodopsin is embedded in the sealed disc membranes, which are isolated and\nelectrically decoupled from the plasma membrane of the outer segment (Fig. 4A) [3, 13]. In\ncontrast, cone discs are direct infoldings of the plasma membrane itself [14].”\n(11) L63-65: The “overshadowed” is confusing here. The only technique mentioned thus far is patch-\nclamp recordings, in which no class of cells should overshadow another. Could you mean in\nthe ERG recording now? If so, it should be stated more clearly. This applies to line 52+ above too–\npresumably recordings in DR and RP must be ERG and not patch-clamp recordings.\n[Reply] Indeed, these recordings refer to ERG recording instead of patch-clamp recording. We have\nclarified this point in the revised manuscript.\nLine 53: “Indeed, a reduction in ERP amplitude has been noted in electroretinography (ERG)\nrecordings from patients with diabetic retinopathy and retinitis pigmentosa [11].”\n(12) L66-70: It would be appropriate to cite some antecedant work here including spectral-domain\nphase microscopy4, self-referenced phase imaging5, and perhaps even phase-based bulk-motion\nestimation6.\n4. Choma MA, Ellerbee AK, Yang C, Creazzo TL, Izatt JA. Spectral-Domain Phase Microscopy. Optics Letters.\n2005;30(10):1162-1164.\n5. Jonnal RS, Kocaoglu OP, Wang Q, Lee S, Miller DT. Phase-Sensitive Imaging of the Outer Retina Using Optical Coherence\nTomography and Adaptive Optics. Biomed Opt Express. 2012;3(1):104-124.\n6. Makita S, Hong Y, Yamanari M, Yatagai T, Yasuno Y. Optical Coherence Angiography. Optics Express. 2006;14(17):7821-\n7840.\n[Reply] We have revised the opening sentences of the third paragraph to include early studies\nusing phase-sensitive OCT measurements.\nLine 68: “Optical coherence tomography (OCT) is a powerful imaging modality in biomedical\noptics that enables non-invasive, depth-resolved imaging with micron-level resolution\n[17]. While the amplitude component of OCT signals reveals microstructural features,\nphase-sensitive measurements allow motion detection with nanometer-scale\nsensitivity [Choma et al., 2005, Makita et al., 2006]. In particular, by monitoring the\nphase difference between two retinal layers [Jonnal et al., 2012], phase-sensitive OCT\nhas detected deformations in photoreceptors associated with metabolic events, such\nas photoisomerization and phototransduction [21-29].”\n(13) L77-79: Most ORG papers state putative osmotic causes for elongation, but a recent paper,\nTomczewski (2025)7, suggests that molecular mechanical forces may play a role too. This should\nprobably be mentioned as well, esp. given the role of inter-disc spacers in the present manuscript’s\ntheory of contraction. That paper is cited earlier, but should be mentioned here in the summary of\nORG hypotheses.\n7. Tomczewski S, Curatolo A, Foik A, et al. Photopic flicker optoretinography captures the light-driven length modulation of\nphotoreceptors during phototransduction. Proceedings of the National Academy of Sciences. 2025;122(7):e2421722122.\ndoi:10.1073/pnas.2421722122.\n[Reply] We have included the hypothesis proposed by Tomczewski et al. as one of the potential\nmechanisms.\nLine 85: “In contrast, the slower expansion of COS was linked to osmotic influx of water,\nswelling of the cone opsin and disc membrane [27, 31], and conformational changes in\nphosphodiesterase (PDE6) during phototransduction [Tomczewski et al., 2025].”\nLine 320: “In COS, the rapid contraction (< 20 ms) is followed by an expansion lasting several\nhundred milliseconds [24], which has been attributed to osmolarity-driven water\ninflux, swelling of cone opsins and disc membranes, and conformational changes in\nPDE6 during phototransduction [27, 29, 31].”\n(14) L118-119: Please provide a citation or explanation justifying segmentation of ROST from RPE\nby using the “top of the thick hyperreflective band.” In histological images, the top of that band\nlooks like a mess of ROST, RPE apical processes, RPE apical surface, phagosomes, and other stuff.\nAlso, it would be good to see a diagram illustrating how much of the thick hyperreflective band\nwas used. This is hinted at in Fig. 1A, but it would be good to see a clear picture (either B- scan or\naxial reflectance profile, i.e., B-scan averaged laterally) of the portion of the thick\nhyperreflective band that was used for ΔOPL measurements.\n[Reply] A recent ORG study in rats suggested that the top of the thick hyperreflective band in the\nouter retina primarily originate from the ROS [Tan et al., 2024]. This is also consistent with findings\nfrom a study comparing SD-OCT images and histology [Berger et al., 2014]. We have added these\nreferences to the revised manuscript. Additionally, an intensity profile has been added to Fig. 1A\nto show the positions of the IS/OS and ROST selected for ΔOPL measurements.\n[Tan et al., 2024] Tan B, Li H, Zhuo Y, et al. Light-evoked deformations in rod photoreceptors, pigment epithelium and\nsubretinal space revealed by prolonged and multilayered optoretinography. Nature Communications. 2024;15(1):5156.\n[Berger et al., 2014] A. Berger, S. Cavallero, E. Dominguez, P. Barbe, M. Simonutti, J.-A. Sahel, F. Sennlaub, W. Raoul, M.\nPaques, and A.-P. Bemelmans, “Spectral-domain optical coherence tomography of the rodent eye: Highlighting layers of the\nouter retina using signal averaging and comparison with histology,” PLOS ONE, vol. 9, no. 5, p. e96494, 2014.\n(15) L118-119: Did the authors use any of the ML classification used by Tan (2024)8? According to\nthat paper, while there was a tendency for pixels exhibiting ROS behavior to lie near the top of the\nhyperreflective band, the depth distributions of the various signal types were broad and\noverlapped significantly. The authors of the present manuscript notice any of the pixels selected\nfor analysis “misbehaving,” i.e., acting like RPE pixels instead of ROST pixels?\n8. Tan B, Li H, Zhuo Y, et al. Light-evoked deformations in rod photoreceptors, pigment epithelium and subretinal space\nrevealed by prolonged and multilayered optoretinography. Nature Communications. 2024;15(1):5156.\ndoi:10.1038/s41467-024-49014-5.\n[Reply] The machine learning classification approach is valuable for sorting signals extracted from\nall pixels, thereby maximizing the SNR of the measured ORG responses. In this study, since the\namplitude of the rapid contraction is relatively large, we simply used pixels in the top of the\nhyperreflective band, where the ORG signal is dominated by the response from ROS. Indeed, the\nhyperreflective band comprises different types of signals, and a recent study revealed movement\nof various outer retinal layers accompanying the rapid contraction of the rod OS [Li et al., 2025].\n[Li et al., 2025] H. Li, C. E. Weiss, V. P. Pandiyan, D. Nanni, T. Liu, P. W. Kung, B. Tan, V. A. Barathi, L. Schmetterer, R.\nSabesan, and T. Ling, “Optoretinography reveals rapid rod photoreceptor movement upon photoisomerization,” bioRxiv, p.\n2025.03.22.644466, 2025.\n(16) L118-119: The authors should state the SNR range of the IS/OS and ROST pixels. This\ndetermines the theoretical phase sensitivity. This is related to the “L402+ and Fig. 1A” comment\nbelow.\n[Reply] The structural image shown in Fig. 1A was averaged across 100 frames to improve the\nvisualization. For a single frame, the SNR for pixels in the IS/OS and ROST is approximately 15 dB\nand 20 dB, respectively. This information has been added to the caption of Fig. 1A.\nCaption of Fig 1A:\n“For a single frame, the SNR for pixels in the IS/OS and ROST is approximately 15 dB\nand 20 dB, respectively.”\n(17) L402+ and Fig. 1A: it would be useful to see some fundamental characteristics of the OCT\nsystem. Fig. 1A indicates an “intensity” range of 35 dB, which corresponds to a linear dynamic\nrange of 32x. Generally intensity is proportional to power/#photons/energy, etc, which means it’s\nproportional to the square of the OCT amplitude. Conventionally, 20log10(amp) is intensity in\ndB, while 10log10(amplitude) is amplitude in dB. It’s important to be specific and detailed about\nthis since the SNR governs phase sensitivity4.\n4. Choma MA, Ellerbee AK, Yang C, Creazzo TL, Izatt JA. Spectral-Domain Phase Microscopy. Optics Letters.\n2005;30(10):1162-1164.\n[Reply] The intensity (dB) in Fig. 1A was calculated as 10×log of the OCT signal intensity, or\n10\nequivalently, 20×log of the OCT signal amplitude. This information has been included in the\n10\ncaption of Fig. 1A.\nCaption of Fig 1A:\n“Intensity (dB) was calculated as 10×log of the OCT signal intensity.”\n10\n(18) L129-130 and Fig. 1C"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my comments satisfactorily.\nHowever, I propose to change the response to comment (17) of reviewer 3:\nThe logarithmic scaling of the OCT signal often leads to confusions as mentioned by the reviewer. The sentence “Intensity (dB) was calculated as 10×log10 of the OCT signal intensity” may as well be misleading since \"OCT signal intensity\" in not clearly defined. The accepted standard is, as used here, 20 Log10 of the absolute value of the Fourier transform the interference spectrum. Hence, the added comment may even be confusing. I would suggest to add to Section IV D the explanation, that OCT signal was calculated by 20 Log10 of the absolute value of the Fourier transform instead of changing the caption of Fig. 1.\nIn response of the comments of all reviewer, the manuscript has significantly improved. It presents an important contribution to the field of ORG.", "reviewer_2": "The authors have adequately addressed all three reviewer's comments. In particular, combining the original Figs. S8 and S9 and moving it to the main manuscript better clarifies the dynamics of the transition pathways described in the paper. I recommend publication.", "reviewer_3": "The authors have addressed my concerns adequately. They have modified figures and added many pieces of important quantitative information. I recommend publication."}, "author_response": ""}]

Decision letter