Biology subjects

Sabesan, R.

Publications and source records attributed to Sabesan, R..

12 recordsLinked to original sources

bioRxiv2026

In vivo elastography of the human retina using light-evoked intrinsic actuation

The biomechanical properties of the retina govern its function, structural integrity, and susceptibility to disease, yet remain difficult to measure in vivo due to the lack of safe, spatially localized mechanical actuation. Here, we introduce a framework for probing retinal biomechanics in the living human eye by leveraging intrinsic optical actuation driven by phototransduction. Using phase-resolved optical coherence tomography with a local phase-referencing approach, we resolved signed, nanometer-scale displacements of the major outer retinal interfaces evoked by light. The resulting deformation field, originating in the photoreceptor outer segment, was distributed across retinal compartments in an eccentricity-dependent manner, with efficient axial transfer in the fovea and attenuated propagation in the parafovea. A hybrid analytical and finite-element framework was developed that retrieved the biomechanical properties of the retinal compartments based on their coordinated deformation and the anatomical variation in retinal structure versus eccentricity. In retinitis pigmentosa, the paradigm enabled the detection of light-evoked deformation in the transition zone despite the loss of native lamination, enabling a functional readout of the vulnerable photoreceptors at the leading edge of degeneration. Together, these results establish intrinsic optical stimulation as a basis for in vivo retinal elastography and enable the non-invasive, quantitative imaging of retinal biomechanics and function in the living human retina.

biophysics↗

bioRxiv2026

Optoretinography in R9AP-bradyopsia reveals the essential role of G-protein signaling in the human cone elongation response

Human cone and rod outer segments exhibit a rapid shrinkage followed by a slower elongation in response to light, forming the basis of the optoretinogram. The molecular basis of this optical assay of photoreceptor function remains incompletely understood. In mouse rods, the elongation response requires transducin, the G-protein -subunit activated in the initial amplifying step of the phototransduction cascade. Here, we measured human cone outer segment responses in subjects with bradyopsia arising from a triple-deletion mutation in R9AP, the anchor and transport protein for the GTPase-activating protein (GAP) RGS9. Immunoprecipitation showed that the mutant R9AP has greatly reduced affinity for RGS9, predictably reducing the level of RGS9 in the outer segment. The bradyopsia subjects elongation responses had normal activation kinetics, amplitude and photosensitivity, but markedly slowed recovery. These results indicate that normal levels of RGS9 are required for deactivation of the cone elongation response, situating the underlying molecular mechanism within the G-protein cascade at or prior to formation of the GAP complex. The recovery of the cone elongation response, measured in a paired-flash paradigm for stimuli isomerizing up to 75% of opsin, revealed that elongation requires a substrate that is depleted and recovers in a bleach-dependent manner. In particular, recovery from the highest bleaching exposure (75%) tracked the time course of cone opsin regeneration, implying that unregenerated cone opsin produces "dark light", known in rods to arise from constitutive activation of G-protein. Taken together, the results from controls and bradyopsia identify the inactive (GDP-bound) transducin complex as the essential substrate for the human cone elongation response. Suppression of the elongation response in the paired-flash paradigm further revealed the complete profile of the initial fast cone outer segment shrinkage response and reinforced its origin in opsin structural changes. Overall, these results identify key mechanistic elements of the optoretinogram and enable its use as a molecularly interpretable, non- invasive assay of cone function in disease and therapeutic response.

neuroscience↗

bioRxiv2025

Optoretinography reveals rapid rod photoreceptor movement upon photoisomerization

Rod photoreceptors are essential for vision under dim light conditions. The onset of rod-mediated vision is marked by the isomerization of rhodopsin. Here we demonstrate that human and rodent rods undergo a minute and rapid contraction of their outer segments immediately upon photoisomerization. The contraction is explained as an electro-mechanical manifestation of the rod early receptor potential generated in the disk membranes, which is challenging to access in electrophysiology. The bleach-strength dependence of the contraction was accounted by a voltage-dependent membrane tension model, developed earlier to explain a similar behavior in cones. The in vivo optical imaging of light-evoked electrical activity in rodent rods was facilitated by an ultrahigh-resolution point-scan optical coherence tomography (OCT) system coupled with unsupervised learning, while in humans, an adaptive optics line-scan OCT facilitated high-speed recordings in individual rods. The non-invasive in vivo optical imaging of rhodopsin activation will have a significant impact on diagnostics and treatment of retinal disease, especially given the vulnerability of rods in inherited and age-related macular degeneration.

bioengineering↗

bioRxiv2025

Local variations in L/M ratio influence the detection and color naming of small spots

The distribution of long (L), middle (M), and short (S) wavelength sensitive cones in the retina determines how different frequencies of incident light are sampled across space and has been hypothesized to influence spatial and color vision. We examined how the detection and color naming of small, short-duration increment stimuli ({lambda} = 543 or 680 nm) depend on the local spectral topography of the cone mosaic. Stimuli were corrected for optical aberrations by an adaptive optics system and targeted to locations in the parafovea where cone spectral types were known. We found that sensitivity to 680 nm light, normalized by sensitivity to 543 nm light, grew with the proportion of L cones at the stimulated locus, though intra- and intersubject variability was considerable. A similar trend was derived from a simple model of the achromatic (L+M) pathway suggesting that small spot detection mainly relies on a non-opponent mechanism. Most stimuli were called achromatic, with red and green responses becoming more common as stimulus intensity increased and as the local L/M ratio became more symmetric. The proximity of S cones to the stimulated region did not influence the likelihood of eliciting a chromatic percept. Our detection data confirm earlier reports that small spot psychophysics can reveal information about local cone topography, and our color naming findings suggest that chromatic sensitivity may improve when the L/M ratio approaches unity.

neuroscience↗

bioRxiv2022

Coarse-scale Optoretinography(CoORG) with extended field-of-view for normative characterization.

Optoretinography (ORG) has the potential to be an effective biomarker for light-evoked retinal activity owing to its sensitive, objective, and precise localization of retinal function and dysfunction. Many ORG implementations have used adaptive optics (AO) to localize activity on a cellular scale. However, the use of AO restricts field-of-view (FOV) to the isoplanatic angle, necessitating the montaging of multiple regions-of-interest to cover an extended field. In addition, subjects with lens opacities, increased eye movements and decreased mobility pose challenges for effective AO operation. Here, we developed a coarse-scale ORG (CoORG) system without AO, which accommodates FOVs up to 5.5 deg. in a single acquisition. The system is based on a line-scan spectral domain OCT with volume rates of up to 32 Hz (16,000 B-frames per second). For acquiring ORGs, 5.5 deg. wide OCT volumes were recorded after dark adaptation and two different stimulus bleaches. The stimulus-evoked optical phase change was calculated from the reflections encasing the cone outer segments and its variation was assessed vs. eccentricity in 12 healthy subjects. The general behavior of {Delta}OPL vs. time mimicked published reports. High trial-to-trial repeatability was observed across subjects and with eccentricity. Comparison of ORG between CoORG and AO-OCT based ORG at 1.5{degrees}, 2.5{degrees}, and 3.5{degrees} eccentricity showed an excellent agreement in the same 2 subjects. The amplitude of the ORG response decreased with increasing eccentricity. The variation of ORG characteristics between subjects and versus eccentricity was well explained by the photon density of the stimulus on the retina and the outer segment length. Overall, the high repeatability and rapid acquisition over an extended field enabled the normative characterization of the cone ORG response in healthy eyes, and provides a promising avenue for translating ORG for widespread clinical application.

bioengineering↗

bioRxiv2022

Characterizing Cone Spectral Classification by Optoretinography

Light propagation in photoreceptor outer segments is affected by photopigment absorption and the phototransduction amplification cascade. Photopigment absorption has been studied using retinal densitometry, while recently, optoretinography (ORG) has provided an avenue to probe changes in outer segment optical path length due to phototransduction. With adaptive optics (AO), both densitometry and ORG have been used for cone spectral classification, based on the differential bleaching signatures of the three cone types. Here, we characterize cone classification by ORG, implemented in an AO line-scan OCT and compare it against densitometry. The cone mosaics of five color normal subjects were classified using ORG showing high probability ([~]0.99), low error (<0.22%), high test-retest reliability ([~]97%) and short imaging durations (< 1 hour). Of these, the cone spectral assignments in two subjects were compared against AOSLO densitometry. High agreement (mean: 91%) was observed between the two modalities in these 2 subjects, with measurements conducted 6-7 years apart. Overall, ORG benefits from higher sensitivity and dynamic range to probe cone photopigments compared to densitometry, and thus provides greater fidelity for cone spectral classification.

neuroscience↗

bioRxiv2022

Shot-noise limited phase-sensitive imaging of moving samples by phase-restoring subpixel motion correction in Fourier-domain optical coherence tomography

Phase-sensitive Fourier-domain optical coherence tomography (FD-OCT) enables in-vivo, label- free imaging of cellular movements with detection sensitivity down to the nanometer scale, and it is widely employed in emerging functional imaging modalities, such as optoretinography (ORG), Doppler OCT, and optical coherence elastography. However, when imaging tissue dynamics in vivo, tissue movement or bulk motion introduces decorrelation noise that compromises motion detection performance, particularly in terms of sensitivity and accuracy. Here, we demonstrate that the motion-related decorrelation noise in FD-OCT can be accurately corrected by restoring the initial sampling points using our proposed Phase-Restoring Subpixel Image Registration (PRESIR) method. Derived from a general FD-OCT model, the PRESIR method enables translational shifting of complex-valued OCT images over arbitrary displacements with subpixel precision, while accurately restoring phase components. Unlike conventional approaches that shift OCT images either in the spatial domain at the pixel level or in the spatial frequency domain for subpixel correction, our method reconstructs OCT images by correcting axial displacement in the spectral domain (k domain) and lateral displacement in the spatial frequency domain. We validated the PRESIR method through simulations, phantom experiments, and in-vivo optoretinography in both rodents and human subjects. Our approach significantly reduced decorrelation noise during the imaging of moving samples, achieving phase sensitivity close to the fundamental limit determined by the signal-to-noise ratio (SNR).

bioengineering↗

bioRxiv2021

Reflective mirror-based line-scan adaptive optics OCT

Line-scan OCT, incorporated with adaptive optics (AO), offers high resolution, speed and sensitivity for imaging retinal structure and function in vivo. Here, we introduce its implementation with reflective mirror-based afocal telescopes, optimized for imaging light-induced retinal activity (optoretinography) and weak retinal reflections at the cellular scale. A non-planar optical design was followed based on previous recommendations with key differences specific to a line-scan geometry. The three beam paths fundamental to an OCT system - illumination/sample, detection, and reference - were modeled in Zemax optical design software to yield theoretically diffraction-limited performance over a 2.2 deg. field-of-view and 1.5 D vergence range at the eyes pupil. The performance for imaging retinal structure was exemplified by cellular-scale visualization of retinal ganglion cells, macrophages, foveal cones, and rods in human observers. The performance for functional imaging was exemplified by resolving the light-evoked optical changes in foveal cone photoreceptors where the spatial resolution was sufficient for cone spectral classification at an eccentricity 0.3 deg. from the foveal center. This enabled the first in vivo demonstration of reduced S-cone (short-wavelength cone) density in the human foveola, thus far observed only in ex vivo histological preparations. Together, the feasibility for high resolution imaging of retinal structure and function demonstrated here holds significant potential for basic science and translational applications.

bioengineering↗

bioRxiv2020

High-speed adaptive optics line-scan OCT for cellular-resolution optoretinography

Optoretinography - the non-invasive, optical imaging of light-induced functional activity in the retina - stands to provide a critical biomarker for testing the safety and efficacy of new therapies as well as their rapid translation to the clinic. Optical phase change in response to light, as readily accessible in phase-resolved OCT, offers a path towards all-optical imaging of retinal function. However, typical human eye motion adversely affects phase stability and precludes the recording of fast light-induced retinal events. Here we introduce a high-speed line-scan spectral domain OCT with adaptive optics (AO), aimed at volumetric imaging and phase-resolved acquisition of retinal responses to light. By virtue of parallel acquisition of an entire retinal cross-section (B-scan) in a single high-speed camera frame, depth-resolved tomograms at speeds up to 16 kHz were achieved with high sensitivity and phase stability. To optimize spectral and spatial resolution, an anamorphic detection paradigm was introduced enabling improved light collection efficiency and signal roll-off compared to traditional methods. The benefits in speed, resolution and sensitivity were exemplified in imaging nanometer-millisecond scale light-induced optical path length changes in cone photoreceptor outer segments. With 660 nm stimuli, individual cone responses readily segregated into three clusters, corresponding to long, middle and short-wavelength cones. Recording such optoretinograms on spatial scales ranging from individual cones, to 100 m-wide retinal patches offers a robust and sensitive biomarker for cone function in health and disease. Furthermore, incorporating this capability into an easy-to-use and ubiquitous diagnostic platform of OCT enables its widespread application to patient care and drug development.

bioengineering↗

bioRxiv2020

The optoretinogram reveals how human photoreceptors deform in response to light

Limited accessibility of retinal neurons to electrophysiology on a cellular scale in-vivo has restricted studies of their signaling to in-vitro preparations and animal models. Physiological changes underlying neural activity are mediated by variations in electrical potential that alter the surface tension of the cell membrane. In addition, physiological processes affect concentration of the cells constituents that results in variation of osmotic pressure. Both these phenomena affect the neurons shape which can be detected using interferometric imaging, thereby enabling non-invasive label-free imaging of physiological activity in-vivo with cellular resolution. Here, we apply high-speed phase-resolved optical coherence tomography in line-field configuration to image the biophysical phenomena associated with phototransduction in human cone photoreceptors in vivo. We demonstrate that individual cones exhibit a biphasic response to light: an early ms-scale fast contraction of the outer segment immediately after the onset of the flash stimulus followed by a gradual (hundreds of ms) expansion. We demonstrate that the contraction can be explained by rapid charge movement accompanying the isomerization of cone opsins, consistent with the early receptor potential observed in the electroretinogram and classical electrophysiology in-vitro. We demonstrate the fidelity of such all-optical recording of light-induced activity in the human retina, namely the optoretinogram, across a range of spatiotemporal scales. This approach incorporates functional evaluation into a routine clinical examination of retinal structure and thus holds enormous potential to serve as a biomarker for early disease diagnosis and monitoring therapeutic efficacy.

neuroscience↗

bioRxiv2020

On mechanisms of light-induced deformations in photoreceptors

Photoreceptors in the retina convert light into electrical signals through a phototransduction cycle that consists of multiple electrical and biochemical events. Phase-resolved optical coherence tomography (pOCT) measurements of the optical path length (OPL) change in the cone photoreceptor outer segments after a light stimulus (optoretinogram) reveal a fast, ms-scale contraction by tens of nm, followed by a slow (hundreds of ms) elongation reaching hundreds of nm. Ultrafast measurements with a line-scan pOCT system show that the contractile response amplitude increases logarithmically with the number of incident photons, and its peak shifts earlier at higher stimulus intensities. We present a model that accounts for these features of the contractile response. Conformational changes in opsins after photoisomerization result in the fractional shift of charge across the disk membrane, leading to a transmembrane voltage change, known as the early receptor potential (ERP). Lateral repulsion of the ions on both sides of the membrane affects its surface tension and leads to its lateral expansion. Since the volume of the disks does not change much on a ms time scale, their lateral expansion leads to an axial contraction of the outer segment. With increasing stimulus intensity and resulting tension, the area expansion coefficient of the disk membrane also increases as thermally-induced fluctuations are pulled flat, resisting further expansion. This results in a logarithmic saturation of the deformation and a peak shift to earlier with brighter stimuli. Slow expansion of the photoreceptors is explained by the influx of water due to osmotic changes during phototransduction. Both effects closely match measurements in healthy human volunteers.

biophysics↗

bioRxiv2018

Sensations from a single M-cone depend on the activity of surrounding S-cones

Color vision requires the activity of cone photoreceptors to be compared in post-receptoral circuitry. Decades of psychophysical measurements have quantified the nature of these comparative interactions on a coarse scale. How such findings generalize to a cellular scale remains unclear. To answer that question, we quantified the influence of surrounding light on the appearance of spots targeted to individual cones. The eyes aberrations were corrected with adaptive optics and retinal position was precisely tracked in real-time to compensate for natural movement. Subjects reported the color appearance of each spot. A majority of L-and M-cones consistently gave rise to the sensation of white, while a smaller group repeatedly elicited hue sensations. When blue sensations were reported they were more likely mediated by M- than L-cones. Blue sensations were elicited from M-cones against a short-wavelength light that preferentially elevated the quantal catch in surrounding S-cones, while stimulation of the same cones against a white background elicited green sensations. In one of two subjects, proximity to S-cones increased the probability of blue reports when M-cones were probed. We propose that M-cone increments excited both green and blue opponent pathways, but the relative activity of neighboring cones favored one pathway over the other.

neuroscience↗