Robust real-time estimation of non-uniform angular velocity and sub-pixel jitter in images captured with resonant scanners.

Images captured with resonant scanners are affected by angular velocity fluctuations that result in image distortion and by poor synchronization between scanning and light detection that creates jitter between image rows. We previously demonstrated that both problems can be mitigated in post-processing by recording the scanner orientation in synchrony with the image capture, followed by data resampling [Opt. Express30, 112 (2022)10.1364/OE.446162]. Here we introduce more robust algorithms for estimation of both angular velocity fluctuation and jitter in the presence of random and deterministic noise. We also show linearization of the scanner oscillation model to reduce calculation times by two orders of magnitude, reaching 65,000 jitter estimations per second when using 2,800 samples per image row, and 500,000 when using only 500 samples, easily supporting real-time generation of jitter-corrected images.

Correction of non-uniform angular velocity and sub-pixel jitter in optical scanning.

Optical scanners are widely used in high-resolution scientific, medical, and industrial devices. The accuracy and precision of these instruments are often limited by angular speed fluctuations due to rotational inertia and by poor synchronization between scanning and light detection, respectively. Here we demonstrate that both problems can be mitigated by recording scanner orientation in synchrony with light detection, followed by data resampling. This approach is illustrated with synthetic and experimental data from a point-scanning microscope with a resonant scanner and a non-resonant scanner. Fitting of the resonant scanner orientation data to a cosine model was used to correct image warping and sampling jitter, as well as to precisely interleave image lines collected during the clockwise and counterclockwise resonant scanner portions of the rotation cycle. Vertical scanner orientation data interpolation was used to correct image distortion due to angular speed fluctuations following abrupt control signal changes.

Correction of resonant optical scanner dynamic aberrations using nodal aberration theory.

The rapid oscillation of galvanometric resonant optical scanners introduces linear astigmatism that degrades transverse resolution, and in confocal systems, also reduces signal [V. Akondi et al., Optica 7, 1506, 2020]. Here, we demonstrate correction of this aberration by tilting reflective or refractive optical elements for a single vergence or a vergence range, with and without the use of an adaptive wavefront corrector such as a deformable mirror. The approach, based on nodal aberration theory, can generate any desired third order aberration that results from tilting or decentering optical surfaces.

Shack-Hartmann wavefront sensor optical dynamic range.

The widely used lenslet-bound definition of the Shack-Hartmann wavefront sensor (SHWS) dynamic range is based on the permanent association between groups of pixels and individual lenslets. Here, we formalize an alternative definition that we term optical dynamic range, based on avoiding the overlap of lenslet images. The comparison of both definitions for Zernike polynomials up to the third order plus spherical aberration shows that the optical dynamic range is larger by a factor proportional to the number of lenslets across the SHWS pupil. Finally, a pre-centroiding algorithm to facilitate lenslet image location in the presence of defocus and astigmatism is proposed. This approach, based on the SHWS image periodicity, is demonstrated using optometric lenses that translate lenslet images outside the projected lenslet boundaries.

Dynamic wavefront distortion in resonant scanners.

Dynamic mirror deformation can substantially degrade the performance of optical instruments using resonant scanners. Here, we evaluate two scanners with resonant frequencies >12kHz with low dynamic distortion. First, we tested an existing galvanometric motor with a novel, to the best of our knowledge, mirror substrate material, silicon carbide, which resonates at 13.8 kHz. This material is stiffer than conventional optical glasses and has lower manufacturing toxicity than beryllium, the stiffest material currently used for this application. Then, we tested a biaxial microelectromechanical (MEMS) scanner with the resonant axis operating at 29.4 kHz. Dynamic deformation measurements show that wavefront aberrations in the galvanometric scanner are dominated by linear oblique astigmatism (90%), while wavefront aberrations in the MEMS scanner are dominated by horizontal coma (30%) and oblique trefoil (27%). In both scanners, distortion amplitude increases linearly with deflection angle, yielding diffraction-limited performance over half of the maximum possible deflection for wavelengths longer than 450 nm and over the full deflection range for wavelengths above 850 nm. Diffraction-limited performance for shorter wavelengths or over larger fractions of the deflection range can be achieved by reducing the beam diameter at the mirror surface. The small dynamic distortion of the MEMS scanner offers a promising alternative to galvanometric resonant scanners with desirable but currently unattainably high resonant frequencies.

Average gradient of Zernike polynomials over polygons.

Wavefront estimation from slope sensor data is often achieved by fitting measured slopes with Zernike polynomial derivatives averaged over the sampling subapertures. Here we discuss how the calculation of these average derivatives can be reduced to one-dimensional integrals of the Zernike polynomials, rather than their derivatives, along the perimeter of each subaperture. We then use this result to derive closed-form expressions for the average Zernike polynomial derivatives over polygonal areas, only requiring evaluation of polynomials at the polygon vertices. Finally, these expressions are applied to simulated Shack-Hartmann wavefront sensors with 7 and 23 fully illuminated lenslets across a circular pupil, with their accuracy and calculation time compared against commonly used integration methods.

Design of two spherical mirror unobscured relay telescopes using nodal aberration theory.

Nodal aberration theory is used to calculate the third-order aberrations that result in image blur for an unobscured modified 4f relay (2f1 + 2f2) formed by two tilted spherical mirrors for objects at infinity (infinite conjugate) and near the front focal plane of the first mirror (finite conjugate). The field-averaged wavefront variance containing only non-rotationally symmetric aberration coefficients is then proposed as an optimization metric. Analytical and ray tracing optimization are demonstrated through sample designs. The particular cases of in-plane and orthogonal folding of the optical axis ray are discussed, followed by an analysis of a modified 2f1 + 2f2 relay in which the distance of the first mirror to the object or pupil is allowed to vary for aberration correction. The sensitivity of the infinite conjugate 2f1 + 2f2 relay to the input marginal ray angle is also examined. Finally, the optimization of multiple conjugate systems through a weighted combination of wavefront variances is proposed.

Accounting for focal shift in the Shack-Hartmann wavefront sensor.

The Shack-Hartmann wavefront sensor samples a beam of light using an array of lenslets, each of which creates an image onto a pixelated sensor. These images translate from their nominal position by a distance proportional to the average wavefront slope over the corresponding lenslet. This principle fails in partially and/or non-uniformly illuminated lenslets when the lenslet array is focused to maximize peak intensity, leading to image centroid bias. Here, we show that this bias is due to the low Fresnel number of the lenslets, which shifts the diffraction focus away from the geometrical focus. We then demonstrate how the geometrical focus can be empirically found by minimizing the bias in partially illuminated lenslets.

Centroid error due to non-uniform lenslet illumination in the Shack-Hartmann wavefront sensor.

Images formed by individual Shack-Hartmann wavefront sensor lenslets are displaced proportionally to the average wavefront slope over their aperture. This principle fails when the lenslet illumination is non-uniform. Here we demonstrate that the resulting error is proportional to the linear component of the illumination intensity, the quadratic wavefront component, and the lenslet size. For illustrative purposes, we compare the error due to centered Gaussian illumination decaying by 30% at the pupil edge against the error due to assuming the wavefront at the lenslet center being equal to the wavefront average across each lenslet. When testing up to ninth-order Zernike polynomial wavefronts and simulating nine lenslets across the pupil, the maximum centroid errors due to non-uniform illumination and sampling are 1.4% and 21%, respectively, and 0.5% and 6.7% when considering 25 lenslets across the pupil in the absence of other sources of error.

CELLULAR IMAGING OF THE TAPETAL-LIKE REFLEX IN CARRIERS OF RPGR-ASSOCIATED RETINOPATHY.

PURPOSE: To examine the features of the tapetal-like reflex (TLR) in female carriers of RPGR-associated retinopathy by means of adaptive optics scanning light ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography. METHODS: Nine molecularly confirmed RPGR carriers and three healthy controls underwent ocular examination and the following retinal imaging modalities: color photography, near-infrared reflectance, fundus autofluorescence, spectral domain optical coherence tomography, and AOSLO. After identifying TLR areas across all imaging modalities, normalized local contrast of outer retinal bands on spectral domain optical coherence tomography was calculated and AOSLO-acquired photoreceptor mosaic analysis was performed. RESULTS: Seven carriers had TLR areas, which colocalized with increased rod photoreceptor reflectivity on confocal AOSLO and reduced cone photoreceptor densities. Parafoveal TLR areas also exhibited reduced local contrast (i.e., increased reflectivity) of the outer retinal bands on spectral domain optical coherence tomography (inner segment ellipsoid zone and outer segment interdigitation zone). Healthy controls did not show TLR. CONCLUSION: The cellular resolution provided by AOSLO affords the characterization of the photoreceptor mosaic in RPGR carriers with a TLR. Features revealed include reduced cone density, increased cone inner segment diameter, and increased rod outer segment reflectivity.

Visible light optical coherence microscopy of the brain with isotropic femtoliter resolution in vivo.

Most flying-spot optical coherence tomography and optical coherence microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single-mode optical fiber. Here we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a water immersion objective on the illumination path while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter ∼0.82 Airy disks), enabling ∼1.1 µm full width at half-maximum (FWHM) transverse resolution. At the same time, a ∼0.9 µm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory and, finally, used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull.

Spatial summation in the human fovea: Do normal optical aberrations and fixational eye movements have an effect?

Psychophysical inferences about the neural mechanisms supporting spatial vision can be undermined by uncertainties introduced by optical aberrations and fixational eye movements, particularly in fovea where the neuronal grain of the visual system is fine. We examined the effect of these preneural factors on photopic spatial summation in the human fovea using a custom adaptive optics scanning light ophthalmoscope that provided control over optical aberrations and retinal stimulus motion. Consistent with previous results, Ricco's area of complete summation encompassed multiple photoreceptors when measured with ordinary amounts of ocular aberrations and retinal stimulus motion. When both factors were minimized experimentally, summation areas were essentially unchanged, suggesting that foveal spatial summation is limited by postreceptoral neural pooling. We compared our behavioral data to predictions generated with a physiologically-inspired front-end model of the visual system, and were able to capture the shape of the summation curves obtained with and without pre-retinal factors using a single postreceptoral summing filter of fixed spatial extent. Given our data and modeling, neurons in the magnocellular visual pathway, such as parasol ganglion cells, provide a candidate neural correlate of Ricco's area in the central fovea.

PHOTORECEPTOR INNER SEGMENT MORPHOLOGY IN BEST VITELLIFORM MACULAR DYSTROPHY.

PURPOSE: To characterize outer retina structure in best vitelliform macular dystrophy (BVMD) and to determine the effect of macular lesions on overlying and adjacent photoreceptors. METHODS: Five individuals with BVMD were followed prospectively with spectral domain optical coherence tomography and confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). The AOSLO cone photoreceptor mosaic images were obtained within and around retinal lesions. Cone density was measured inside and outside lesions. In 2 subjects, densities were compared with published measurements acquired ∼2.5 years before. One subject was imaged 3 times over a 5-month period. RESULTS: The AOSLO imaging demonstrated that photoreceptor morphology within BVMD retinal lesions was highly variable depending on the disease stage, with photoreceptor structure present even in advanced disease. The AOSLO imaging was repeatable even in severe disease over short-time and long-time intervals. Photoreceptor density was normal in retinal areas immediately adjacent to lesions and stable over ∼2.5 years. Mobile disk-like structures possibly representing subretinal macrophages were also observed. CONCLUSION: Combined confocal and nonconfocal split-detector AOSLO imaging reveals substantial variability within clinical lesions in all stages of BVMD. Longitudinal cellular photoreceptor imaging could prove a powerful tool for understanding disease progression and monitoring emerging therapeutic treatment response in inherited degenerations such as BVMD.

REPEATABILITY AND LONGITUDINAL ASSESSMENT OF FOVEAL CONE STRUCTURE IN CNGB3-ASSOCIATED ACHROMATOPSIA.

PURPOSE: Congenital achromatopsia is an autosomal recessive disease causing substantial reduction or complete absence of cone function. Although believed to be a relatively stationary disorder, questions remain regarding the stability of cone structure over time. In this study, the authors sought to assess the repeatability of and examine longitudinal changes in measurements of central cone structure in patients with achromatopsia. METHODS: Forty-one subjects with CNGB3-associated achromatopsia were imaged over a period of between 6 and 26 months using optical coherence tomography and adaptive optics scanning light ophthalmoscopy. Outer nuclear layer (ONL) thickness, ellipsoid zone (EZ) disruption, and peak foveal cone density were assessed. RESULTS: ONL thickness increased slightly compared with baseline (0.184 µm/month, P = 0.02). The EZ grade remained unchanged for 34/41 subjects. Peak foveal cone density did not significantly change over time (mean change 1% per 6 months, P = 0.126). CONCLUSION: Foveal cone structure showed little or no change in this group of subjects with CNGB3-associated achromatopsia. Over the time scales investigated (6-26 months), achromatopsia seems to be a structurally stable condition, although longer-term follow-up is needed. These data will be useful in assessing foveal cone structure after therapeutic intervention.

Noninvasive imaging of the thirteen-lined ground squirrel photoreceptor mosaic.

Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel (Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale.

ASSESSING PHOTORECEPTOR STRUCTURE ASSOCIATED WITH ELLIPSOID ZONE DISRUPTIONS VISUALIZED WITH OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To compare images of photoreceptor layer disruptions obtained with optical coherence tomography (OCT) and adaptive optics scanning light ophthalmoscopy (AOSLO) in a variety of pathologic states. METHODS: Five subjects with photoreceptor ellipsoid zone disruption as per OCT and clinical diagnoses of closed-globe blunt ocular trauma (n = 2), macular telangiectasia type 2 (n = 1), blue-cone monochromacy (n = 1), or cone-rod dystrophy (n = 1) were included. Images were acquired within and around photoreceptor lesions using spectral domain OCT, confocal AOSLO, and split-detector AOSLO. RESULTS: There were substantial differences in the extent and appearance of the photoreceptor mosaic as revealed by confocal AOSLO, split-detector AOSLO, and spectral domain OCT en face view of the ellipsoid zone. CONCLUSION: Clinically available spectral domain OCT, viewed en face or as B-scan, may lead to misinterpretation of photoreceptor anatomy in a variety of diseases and injuries. This was demonstrated using split-detector AOSLO to reveal substantial populations of photoreceptors in areas of no, low, or ambiguous ellipsoid zone reflectivity with en face OCT and confocal AOSLO. Although it is unclear if these photoreceptors are functional, their presence offers hope for therapeutic strategies aimed at preserving or restoring photoreceptor function.

Longitudinal imaging of microvascular remodelling in proliferative diabetic retinopathy using adaptive optics scanning light ophthalmoscopy.

PURPOSE: To characterise longitudinal changes in the retinal microvasculature of type 2 diabetes mellitus (T2DM) as exemplified in a patient with proliferative diabetic retinopathy (PDR) using an adaptive optics scanning light ophthalmoscope (AOSLO). METHODS: A 35-year-old T2DM patient with PDR treated with scatter pan-retinal photocoagulation at the inferior retina 1 day prior to initial AOSLO imaging along with a 24-year-old healthy control were imaged in this study. AOSLO vascular structural and perfusion maps were acquired at four visits over a 20-week period. Capillary diameter and microaneurysm area changes were measured on the AOSLO structural maps. Imaging repeatability was established using longitudinal imaging of microvasculature in the healthy control. RESULTS: Capillary occlusion and recanalisation, capillary dilatation, resolution of local retinal haemorrhage, capillary hairpin formation, capillary bend formation, microaneurysm formation, progression and regression were documented over time in a region 2° superior to the fovea in the PDR patient. An identical microvascular network with same capillary diameter was observed in the control subject over time. CONCLUSIONS: High-resolution serial AOSLO imaging enables in vivo observation of vasculopathic changes seen in diabetes mellitus. The implications of this methodology are significant, providing the opportunity for studying the dynamics of the pathological process, as well as the possibility of identifying highly sensitive and non-invasive biomarkers of end organ damage and response to treatment.

FELLOW EYE CHANGES IN PATIENTS WITH NONISCHEMIC CENTRAL RETINAL VEIN OCCLUSION: Assessment of Perfused Foveal Microvascular Density and Identification of Nonperfused Capillaries.

PURPOSE: Eyes fellow to nonischemic central retinal vein occlusion (CRVO) were examined for abnormalities, which might explain their increased risk for future occlusion, using adaptive optics scanning light ophthalmoscope fluorescein angiography. METHODS: Adaptive optics scanning light ophthalmoscope fluorescein angiography foveal microvascular densities were calculated. Nonperfused capillaries adjacent to the foveal avascular zone were identified. Spectral domain optical coherence tomography, ultrawide field fluorescein angiographies, and microperimetry were also performed. RESULTS: Ten fellow eyes of nine nonischemic CRVO and 1 nonischemic hemi-CRVO subjects and four affected eyes of three nonischemic CRVO and one nonischemic hemi-CRVO subjects were imaged. Ninety percent of fellow eyes and 100% of affected eyes demonstrated at least 1 nonperfused capillary compared with 31% of healthy eyes. Fellow eye microvascular density (35 ± 3.6 mm(-1)) was significantly higher than that of affected eyes (25 ± 5.2 mm(-1)) and significantly lower than that of healthy eyes (42 ± 4.2 mm(-1)). Compared with healthy controls, spectral domain optical coherence tomography thicknesses showed no significant difference, whereas microperimetry and 2/9 ultrawide field fluorescein angiography revealed abnormalities in fellow eyes. CONCLUSION: Fellow eye changes detectable on adaptive optics scanning light ophthalmoscope fluorescein angiography reflect subclinical pathology difficult to detect using conventional imaging technologies. These changes may help elucidate the pathogenesis of nonischemic CRVO and help identify eyes at increased risk of future occlusion.

Retinal structure and function in achromatopsia: implications for gene therapy.

PURPOSE: To characterize retinal structure and function in achromatopsia (ACHM) in preparation for clinical trials of gene therapy. DESIGN: Cross-sectional study. PARTICIPANTS: Forty subjects with ACHM. METHODS: All subjects underwent spectral domain optical coherence tomography (SD-OCT), microperimetry, and molecular genetic testing. Foveal structure on SD-OCT was graded into 5 distinct categories: (1) continuous inner segment ellipsoid (ISe), (2) ISe disruption, (3) ISe absence, (4) presence of a hyporeflective zone (HRZ), and (5) outer retinal atrophy including retinal pigment epithelial loss. Foveal and outer nuclear layer (ONL) thickness was measured and presence of hypoplasia determined. MAIN OUTCOME MEASURES: Photoreceptor appearance on SD-OCT imaging, foveal and ONL thickness, presence of foveal hypoplasia, retinal sensitivity and fixation stability, and association of these parameters with age and genotype. RESULTS: Forty subjects with a mean age of 24.9 years (range, 6-52 years) were included. Disease-causing variants were found in CNGA3 (n = 18), CNGB3 (n = 15), GNAT2 (n = 4), and PDE6C (n = 1). No variants were found in 2 individuals. In all, 22.5% of subjects had a continuous ISe layer at the fovea, 27.5% had ISe disruption, 20% had an absent ISe layer, 22.5% had an HRZ, and 7.5% had outer retinal atrophy. No significant differences in age (P = 0.77), mean retinal sensitivity (P = 0.21), or fixation stability (P = 0.34) across the 5 SD-OCT categories were evident. No correlation was found between age and foveal thickness (P = 0.84) or between age and foveal ONL thickness (P = 0.12). CONCLUSIONS: The lack of a clear association of disruption of retinal structure or function in ACHM with age suggests that the window of opportunity for intervention by gene therapy is wider in some individuals than previously indicated. Therefore, the potential benefit for a given subject is likely to be better predicted by specific measurement of photoreceptor structure rather than simply by age. The ability to directly assess cone photoreceptor preservation with SD-OCT and/or adaptive optics imaging is likely to prove invaluable in selecting subjects for future trials and measuring the trials' impact.

Visualization of retinal vascular structure and perfusion with a nonconfocal adaptive optics scanning light ophthalmoscope.

Imaging of the retinal vascular structure and perfusion was explored by confocal illumination and nonconfocal detection in an adaptive optics scanning light ophthalmoscope (AOSLO), as an extension of the work by Chui et al. [Biomed. Opt. Express 3, 2537 (2012)]. Five different detection schemes were evaluated at multiple retinal locations: circular mask, annular mask, circular mask with filament, knife-edge, and split-detector. Given the superior image contrast in the reflectance and perfusion maps, the split-detection method was further tested using pupil apodization, polarized detection, and four different wavelengths. None of these variations provided noticeable contrast improvement. The noninvasive visualization of capillary flow and structure provided by AOSLO split-detection shows great promise for studying ocular and systemic conditions that affect the retinal vasculature.