Human cone elongation responses can be explained by photoactivated cone opsin and membrane swelling and osmotic response to phosphate produced by RGS9-catalyzed GTPase.

Human cone outer segment (COS) length changes in response to stimuli bleaching up to 99% of L- and M-cone opsins were measured with high resolution, phase-resolved optical coherence tomography (OCT). Responses comprised a fast phase (∼5 ms), during which COSs shrink, and two slower phases (1.5 s), during which COSs elongate. The slower components saturated in amplitude (∼425 nm) and initial rate (∼3 nm ms-1) and are well described over the 200-fold bleaching range as the sum of two exponentially rising functions with time constants of 80 to 90 ms (component 1) and 1,000 to 1,250 ms (component 2). Measurements with adaptive optics reflection densitometry revealed component 2 to be linearly related to cone pigment bleaching, and the hypothesis is proposed that it arises from cone opsin and disk membrane swelling triggered by isomerization and rate-limited by chromophore hydrolysis and its reduction to membrane-localized all-trans retinol. The light sensitivity and kinetics of component 1 suggested that the underlying mechanism is an osmotic response to an amplified soluble by-product of phototransduction. The hypotheses that component 1 corresponds to G-protein subunits dissociating from the membrane, metabolites of cyclic guanosine monophosphate (cGMP) hydrolysis, or by-products of activated guanylate cyclase are rejected, while the hypothesis that it corresponds to phosphate produced by regulator of G-protein signaling 9 (RGS9)-catalyzed hydrolysis of guanosine triphosphate (GTP) in G protein-phosphodiesterase complexes was found to be consistent with the results. These results provide a basis for the assessment with optoretinography of phototransduction in individual cone photoreceptors in health and during disease progression and therapeutic interventions.

Unique yellow shifts for small and brief stimuli in the central retina.

The spectral locus of unique yellow was determined for flashes of different sizes (<11 arcmin) and durations (<500 ms) presented in and near the fovea. An adaptive optics scanning laser ophthalmoscope was used to minimize the effects of higher-order aberrations during simultaneous stimulus delivery and retinal imaging. In certain subjects, parafoveal cones were classified as L, M, or S, which permitted the comparison of unique yellow measurements with variations in local L/M ratios within and between observers. Unique yellow shifted to longer wavelengths as stimulus size or duration was reduced. This effect is most pronounced for changes in size and more apparent in the fovea than in the parafovea. The observed variations in unique yellow are not entirely predicted from variations in L/M ratio and therefore implicate neural processes beyond photoreception.

Cortical distance unifies the extent of parafoveal contour interactions.

It is well known that crowding, the disruptive influence of flanking items on identification of targets, is the primary limiting factor to object identification in the periphery, while limits in the fovea are more determined by the ability to resolve individual items. Whether this is a dichotomous or merely a quantitative difference, and the transition between these two regimes, has remained unexplained. Here, using an adaptive optics system for optimal control of optical and stimulus factors, we measured threshold acuity for identification of Tumbling Es flanked by bars at a variety of flanker spacings and eight eccentricities in the parafovea. Thresholds at each eccentricity were influenced by resolution, contour interaction, and a saturating pedestal effect. When target-flanker spacing was plotted in terms of cortical distance, a single canonical clipped-line fit unified the resultant curves. The critical spacing for letters flanked by bars was found to be 1.3 to 1.5 cortical millimeters, corresponding to approximately 0.1*E outside the fovea.

Correcting intra-volume distortion for AO-OCT using 3D correlation based registration.

Adaptive optics (AO) based ophthalmic imagers, such as scanning laser ophthalmoscopes (SLO) and optical coherence tomography (OCT), are used to evaluate the structure and function of the retina with high contrast and resolution. Fixational eye movements during a raster-scanned image acquisition lead to intra-frame and intra-volume distortion, resulting in an inaccurate reproduction of the underlying retinal structure. For three-dimensional (3D) AO-OCT, segmentation-based and 3D correlation based registration methods have been applied to correct eye motion and achieve a high signal-to-noise ratio registered volume. This involves first selecting a reference volume, either manually or automatically, and registering the image/volume stream against the reference using correlation methods. However, even within the chosen reference volume, involuntary eye motion persists and affects the accuracy with which the 3D retinal structure is finally rendered. In this article, we introduced reference volume distortion correction for AO-OCT using 3D correlation based registration and demonstrate a significant improvement in registration performance via a few metrics. Conceptually, the general paradigm follows that developed previously for intra-frame distortion correction for 2D raster-scanned images, as in an AOSLO, but extended here across all three spatial dimensions via 3D correlation analyses. We performed a frequency analysis of eye motion traces before and after intra-volume correction and revealed how periodic artifacts in eye motion estimates are effectively reduced upon correction. Further, we quantified how the intra-volume distortions and periodic artifacts in the eye motion traces, in general, decrease with increasing AO-OCT acquisition speed. Overall, 3D correlation based registration with intra-volume correction significantly improved the visualization of retinal structure and estimation of fixational eye movements.

Effect of cone spectral topography on chromatic detection sensitivity.

The spatial and spectral topography of the cone mosaic set the limits for detection and discrimination of chromatic sinewave gratings. Here, we sought to compare the spatial characteristics of mechanisms mediating hue perception against those mediating chromatic detection in individuals with known spectral topography and with optical aberrations removed with adaptive optics. Chromatic detection sensitivity in general exceeded previous measurements and decreased monotonically for increasingly skewed cone spectral compositions. The spatial grain of hue perception was significantly coarser than chromatic detection, consistent with separate neural mechanisms for color vision operating at different spatial scales.

Habitual higher order aberrations affect Landolt but not Vernier acuity.

To assess whether the eye's optical imperfections are relevant for hyperacute vision, we measured ocular wave aberrations, visual hyperacuity, and acuity thresholds in 31 eyes of young adults. Although there was a significant positive correlation between the subjects' performance in Vernier- and Landolt-optotype acuity tasks, we found clear differences in how far both acuity measures correlate with the eyes' optics. Landolt acuity thresholds were significantly better in eyes with low higher order aberrations and high visual Strehl ratios (r2 = 0.22, p = 0.009), and significantly positively correlated with axial length (r2 = 0.15, p = 0.03). A retinal image quality metric, calculated as two-dimensional correlation between perfect and actual retinal image, was also correlated with Landolt acuity thresholds (r2 = 0.27, p = 0.003). No such correlations were found with Vernier acuity performance (r2 < 0.03, p > 0.3). Based on these results, hyperacuity thresholds are, contrary to resolution acuity, not affected by higher order aberrations of the eye.

Spatiochromatic Interactions between Individual Cone Photoreceptors in the Human Retina.

A remarkable feature of human vision is that the retina and brain have evolved circuitry to extract useful spatial and spectral information from signals originating in a photoreceptor mosaic with trichromatic constituents that vary widely in their relative numbers and local spatial configurations. A critical early transformation applied to cone signals is horizontal-cell-mediated lateral inhibition, which imparts a spatially antagonistic surround to individual cone receptive fields, a signature inherited by downstream neurons and implicated in color signaling. In the peripheral retina, the functional connectivity of cone inputs to the circuitry that mediates lateral inhibition is not cone-type specific, but whether these wiring schemes are maintained closer to the fovea remains unsettled, in part because central retinal anatomy is not easily amenable to direct physiological assessment. Here, we demonstrate how the precise topography of the long (L)-, middle (M)-, and short (S)-wavelength-sensitive cones in the human parafovea (1.5° eccentricity) shapes perceptual sensitivity. We used adaptive optics microstimulation to measure psychophysical detection thresholds from individual cones with spectral types that had been classified independently by absorptance imaging. Measured against chromatic adapting backgrounds, the sensitivities of L and M cones were, on average, receptor-type specific, but individual cone thresholds varied systematically with the number of preferentially activated cones in the immediate neighborhood. The spatial and spectral patterns of these interactions suggest that interneurons mediating lateral inhibition in the central retina, likely horizontal cells, establish functional connections with L and M cones indiscriminately, implying that the cone-selective circuitry supporting red-green color vision emerges after the first retinal synapse.SIGNIFICANCE STATEMENT We present evidence for spatially antagonistic interactions between individual, spectrally typed cones in the central retina of human observers using adaptive optics. Using chromatic adapting fields to modulate the relative steady-state activity of long (L)- and middle (M)-wavelength-sensitive cones, we found that single-cone detection thresholds varied predictably with the spectral demographics of the surrounding cones. The spatial scale and spectral pattern of these photoreceptor interactions were consistent with lateral inhibition mediated by retinal horizontal cells that receive nonselective input from L and M cones. These results demonstrate a clear link between the neural architecture of the visual system inputs-cone photoreceptors-and visual perception and have implications for the neural locus of the cone-specific circuitry supporting color vision.

Eye-tracking technology for real-time monitoring of transverse chromatic aberration.

Objective measurements of transverse chromatic aberration (TCA) between two or more wavelengths with an adaptive optics scanning laser ophthalmoscope (AOSLO) are very accurate, but frequent measurements are impractical in many experimental settings. Here, we demonstrate a pupil tracker that can accurately measure relative changes in TCA that are caused by small shifts in the pupil relative to the AOSLO imaging beam. Corrections for TCA caused by these shifts improve the measurement of TCA as a function of eccentricity, revealing a strong linear relationship. We propose that pupil tracking be integrated into AOSLO systems, where robust and unobtrusive control of TCA is required.

Transverse chromatic aberration across the visual field of the human eye.

The purpose of this study was to measure the transverse chromatic aberration (TCA) across the visual field of the human eye objectively. TCA was measured at horizontal and vertical field angles out to ±15° from foveal fixation in the right eye of four subjects. Interleaved retinal images were taken at wavelengths 543 nm and 842 nm in an adaptive optics scanning laser ophthalmoscope (AOSLO). To obtain true measures of the human eye's TCA, the contributions of the AOSLO system's TCA were measured using an on-axis aligned model eye and subtracted from the ocular data. The increase in TCA was found to be linear with eccentricity, with an average slope of 0.21 arcmin/degree of visual field angle (corresponding to 0.41 arcmin/degree for 430 nm to 770 nm). The absolute magnitude of ocular TCA varied between subjects, but was similar to the resolution acuity at 10° in the nasal visual field, encompassing three to four cones. Therefore, TCA can be visually significant. Furthermore, for high-resolution imaging applications, whether visualizing or stimulating cellular features in the retina, it is important to consider the lateral displacements between wavelengths and the variation in blur over the visual field.

Effects of Neural Adaptation to Habitual Spherical Aberration on Depth of Focus.

We investigated how long-term visual experience with habitual spherical aberration (SA) influences subjective depth of focus (DoF). Nine healthy cycloplegic eyes with habitual SAs of different signs and magnitudes were enrolled. An adaptive optics (AO) visual simulator was used to measure through-focus high-contrast visual acuity after correcting all monochromatic aberrations and imposing +0.5 µm and -0.5 µm SAs for a 6-mm pupil. The positive (n=6) and negative (n=3) SA groups ranged from 0.17 to 0.8 µm and from -1.2 to -0.12 µm for a 6-mm pupil, respectively. For the positive habitual SA group, the median DoF with positive AO-induced SA (2.18D) was larger than that with negative AO-induced SA (1.91D); for the negative habitual SA group, a smaller DoF was measured with positive AO-induced SA (1.81D) than that with negative AO-induced SA (2.09D). The difference in the DoF of individual participants between the induced positive and negative SA groups showed a quadratic relationship with the habitual SA. Subjective DoF tended to be larger when the induced SA in terms of the sign and magnitude was closer to the participant's habitual SA, suggesting the importance of considering the habitual SA when applying the extended DoF method using optical or surgical procedures.

Wavefront-guided scleral lens prosthetic device for keratoconus.

PURPOSE: To investigate the feasibility of correcting ocular higher order aberrations (HOAs) in keratoconus (KC) using wavefront-guided optics in a scleral lens prosthetic device (SLPD). METHODS: Six advanced KC patients (11 eyes) were fitted with an SLPD with conventional spherical optics. A custom-made Shack-Hartmann wavefront sensor was used to measure aberrations through a dilated pupil wearing the SLPD. The position of SLPD, that is, horizontal and vertical decentration relative to the pupil and rotation were measured and incorporated into the design of the wavefront-guided optics for the customized SLPD. A submicron-precision lathe created the designed irregular profile on the front surface of the device. The residual aberrations of the same eyes wearing the SLPD with wavefront-guided optics were subsequently measured. Visual performance with natural mesopic pupil was compared between SLPDs having conventional spherical and wavefront-guided optics by measuring best-corrected high-contrast visual acuity and contrast sensitivity. RESULTS: Root mean square of HOA in the 11 eyes wearing conventional SLPD with spherical optics was 1.17 ± 0.57 µm for a 6-mm pupil. Higher order aberrations were effectively corrected by the customized SLPD with wavefront-guided optics, and root mean square was reduced 3.1 times on average to 0.37 ± 0.19 µm for the same pupil. This correction resulted in significant improvement of 1.9 lines in mean visual acuity (p < 0.05). Contrast sensitivity was also significantly improved by factors of 2.4, 1.8, and 1.4 on average for 4, 8, and 12 cycles/degree, respectively (p < 0.05 for all frequencies). Although the residual aberration was comparable to that of normal eyes, the average visual acuity in logMAR with the customized SLPD was 0.21, substantially worse than normal acuity. CONCLUSIONS: The customized SLPD with wavefront-guided optics corrected the HOA of advanced KC patients to normal levels and improved their vision significantly.

Introduction to the Feature Issue on Adaptive Optics for Biomedical Applications.

The guest editors introduce a feature issue commemorating the 25th anniversary of adaptive optics in biomedical research.

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 ΔOPL 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°, 2.5°, and 3.5° 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.

Improvement of neural contrast sensitivity after long-term adaptation in pseudophakic eyes.

An adaptive optics (AO) system was used to investigate the effect of long-term neural adaptation to the habitual optical profile on neural contrast sensitivity in pseudophakic eyes after the correction of all aberrations, defocus, and astigmatism. Pseudophakic eyes were assessed at 4 and 8 months postoperatively for changes in visual performance. Visual benefit was observed in all eyes at all spatial frequencies after AO correction. The average visual benefit across spatial frequencies was higher in the pseudophakic group (3.31) at 4 months postoperatively compared to the normal group (2.41). The average contrast sensitivity after AO correction in the pseudophakic group improved by a factor of 1.73 between 4 and 8 months postoperatively. Contrast sensitivity in pseudophakic eyes was poorer, which could be attributed to long-term adaptation to the habitual optical profiles before the cataract surgery, in conjunction with age-related vision loss. Improved visual performance in pseudophakic eyes suggests that the aged neural system can be re-adapted for altered ocular optics.

Interferometric imaging of thermal expansion for temperature control in retinal laser therapy.

Precise control of the temperature rise is a prerequisite for proper photothermal therapy. In retinal laser therapy, the heat deposition is primarily governed by the melanin concentration, which can significantly vary across the retina and from patient to patient. In this work, we present a method for determining the optical and thermal properties of layered materials, directly applicable to the retina, using low-energy laser heating and phase-resolved optical coherence tomography (pOCT). The method is demonstrated on a polymer-based tissue phantom heated with a laser pulse focused onto an absorbing layer buried below the phantom's surface. Using a line-scan spectral-domain pOCT, optical path length changes induced by the thermal expansion were extracted from sequential B-scans. The material properties were then determined by matching the optical path length changes to a thermo-mechanical model developed for fast computation. This method determined the absorption coefficient with a precision of 2.5% and the temperature rise with a precision of about 0.2°C from a single laser exposure, while the peak did not exceed 8°C during 1 ms pulse, which is well within the tissue safety range and significantly more precise than other methods.

Suprathreshold Contrast Perception Is Altered by Long-term Adaptation to Habitual Optical Blur.

Purpose: To investigate whether visual experience with habitual blur alters the neural processing of suprathreshold contrast in emmetropic and highly aberrated eyes. Methods: A large stroke adaptive optics system was used to correct ocular aberrations. Contrast constancy was assessed psychophysically in emmetropic and keratoconic eyes using a contrast matching paradigm. Participants adjusted the contrasts of gratings at various spatial frequencies to match the contrast perception of a reference grating at 4 c/deg. Matching was done both with fully corrected and uncorrected ocular aberrations. Optical correction allowed keratoconus patients to perceive high spatial frequencies that they have not experienced for some time. Results: Emmetropic observers exhibited contrast constancy both with their native aberrations and when their aberrations were corrected. Keratoconus patients exhibited contrast constancy with their uncorrected, native optics but they did not exhibit constancy during adaptive optics correction. Instead. they exhibited striking underconstancy: they required more contrast at high spatial frequencies than the contrast of the 4-c/deg stimulus to make them seem to have the same contrast. Conclusions: The presence of contrast constancy in emmetropes and keratoconus patients viewing with their native optics suggests that they have learned to amplify neural signals to offset the effects of habitual optical aberrations. The fact that underconstancy was observed in keratoconus patients when their optics were corrected suggests that they were unable to learn the appropriate neural amplification because they did not have experience with fine spatial detail. These results show that even adults can learn neural amplification to counteract the effects of their own optical aberrations.

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 optical coherence tomography (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 AO-scanning laser opthalmoscope densitometry. High agreement (mean: 91%) was observed between the two modalities in these two 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.

Reflective mirror-based line-scan adaptive optics OCT for imaging retinal structure and function.

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 eye's 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.

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. In addition, recording fast light-induced retinal events necessitates high-speed acquisition. 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 µm-wide retinal patches offers a robust and sensitive biomarker for cone function in health and disease.

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

Photoreceptors initiate vision by converting photons to electrical activity. The onset of the phototransduction cascade is marked by the isomerization of photopigments upon light capture. We revealed that the onset of phototransduction is accompanied by a rapid (<5 ms), nanometer-scale electromechanical deformation in individual human cone photoreceptors. Characterizing this biophysical phenomenon associated with phototransduction in vivo was enabled by high-speed phase-resolved optical coherence tomography in a line-field configuration that allowed sufficient spatiotemporal resolution to visualize the nanometer/millisecond-scale light-induced shape change in photoreceptors. The deformation was explained as the optical manifestation of electrical activity, caused due to rapid charge displacement following isomerization, resulting in changes of electrical potential and surface tension within the photoreceptor disc membranes. These all-optical recordings of light-induced activity in the human retina constitute an optoretinogram and hold remarkable potential to reveal the biophysical correlates of neural activity in health and disease.