Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.

Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

Probing Computation in the Primate Visual System at Single-Cone Resolution.

Daylight vision begins when light activates cone photoreceptors in the retina, creating spatial patterns of neural activity. These cone signals are then combined and processed in downstream neural circuits, ultimately producing visual perception. Recent technical advances have made it possible to deliver visual stimuli to the retina that probe this processing by the visual system at its elementary resolution of individual cones. Physiological recordings from nonhuman primate retinas reveal the spatial organization of cone signals in retinal ganglion cells, including how signals from cones of different types are combined to support both spatial and color vision. Psychophysical experiments with human subjects characterize the visual sensations evoked by stimulating a single cone, including the perception of color. Future combined physiological and psychophysical experiments focusing on probing the elementary visual inputs are likely to clarify how neural processing generates our perception of the visual world.

Cone-Opponent Ganglion Cells in the Primate Fovea Tuned to Noncardinal Color Directions.

A long-standing question in vision science is how the three cone photoreceptor types-long (L), medium (M), and short (S) wavelength sensitive-combine to generate our perception of color. Hue perception can be described along two opponent axes: red-green and blue-yellow. Psychophysical measurements of color appearance indicate that the cone inputs to the red-green and blue-yellow opponent axes are M vs. L + S and L vs. M + S, respectively. However, the "cardinal directions of color space" revealed by psychophysical measurements of color detection thresholds following adaptation are L vs. M and S vs. L + M. These cardinal directions match the most common cone-opponent retinal ganglion cells (RGCs) in the primate retina. Accordingly, the cone opponency necessary for color appearance is thought to be established in the cortex. While neurons with the appropriate M vs. L + S and L vs. M + S opponency have been reported in the retina and lateral geniculate nucleus, their existence continues to be debated. Resolving this long-standing debate is necessary because a complete account of the cone opponency in the retinal output is critical for understanding how downstream neural circuits process color. Here, we performed adaptive optics calcium imaging to noninvasively measure foveal RGC light responses in the living Macaca fascicularis eye. We confirm the presence of L vs. M + S and M vs. L + S neurons with noncardinal cone opponency and demonstrate that cone-opponent signals in the retinal output are more diverse than classically thought.

Binocular mirror-symmetric microsaccadic sampling enables Drosophila hyperacute 3D vision.

SignificanceTo move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes' optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes' whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies' depth-perception dynamics, limits, and visual behaviors.

Adaptive Gap-Tunable Surface-Enhanced Raman Spectroscopy.

Gap plasmon (GP) resonance in static surface-enhanced Raman spectroscopy (SERS) structures is generally too narrow and not tunable. Here, we present an adaptive gap-tunable SERS device to selectively enhance and modulate different vibrational modes via active flexible Au nanogaps, with adaptive optical control. The tunability of GP resonance is up to ∼1200 cm-1 by engineering gap width, facilitated by mechanical bending of a polyethylene terephthalate substrate. We confirm that the tuned GP resonance selectively enhances different Raman spectral regions of the molecules. Additionally, we dynamically control the SERS intensity through the wavefront shaping of excitation beams. Furthermore, we demonstrate simulation results, exhibiting the mechanical and optical properties of a one-dimensional flexible nanogap and their advantage in high-speed biomedical sensing. Our work provides a unique approach for observing and controlling the enhanced chemical responses with dynamic tunability.

Controlling cantilevered adaptive X-ray mirrors.

Modeling the behavior of a prototype cantilevered X-ray adaptive mirror (held from one end) demonstrates its potential for use on high-performance X-ray beamlines. Similar adaptive mirrors are used on X-ray beamlines to compensate optical aberrations, control wavefronts and tune mirror focal distances at will. Controlled by 1D arrays of piezoceramic actuators, these glancing-incidence mirrors can provide nanometre-scale surface shape adjustment capabilities. However, significant engineering challenges remain for mounting them with low distortion and low environmental sensitivity. Finite-element analysis is used to predict the micron-scale full actuation surface shape from each channel and then linear modeling is applied to investigate the mirrors' ability to reach target profiles. Using either uniform or arbitrary spatial weighting, actuator voltages are optimized using a Moore-Penrose matrix inverse, or pseudoinverse, revealing a spatial dependence on the shape fitting with increasing fidelity farther from the mount.

Absolute retinal blood flow in healthy eyes and in eyes with retinal vein occlusion.

PURPOSE: To measure non-invasively retinal venous blood flow (RBF) in healthy subjects and patients with retinal venous occlusion (RVO). METHODS: The prototype named AO-LDV (Adaptive Optics Laser Doppler Velocimeter), which combines a new absolute laser Doppler velocimeter with an adaptive optics fundus camera (rtx1, Imagine Eyes®, Orsay, France), was studied for the measurement of absolute RBF as a function of retinal vessel diameters and simultaneous measurement of red blood cell velocity. RBF was measured in healthy subjects (n = 15) and patients with retinal venous occlusion (RVO, n = 6). We also evaluated two softwares for the measurement of retinal vessel diameters: software 1 (automatic vessel detection, profile analysis) and software 2 (based on the use of deep neural networks for semantic segmentation of vessels, using a M2u-Net architecture). RESULTS: Software 2 provided a higher rate of automatic retinal vessel measurement (99.5 % of 12,320 AO images) than software 1 (64.9 %) and wider measurements (75.5 ± 15.7 µm vs 70.9 ± 19.8 µm, p < 0.001). For healthy subjects (n = 15), all the retinal veins in one eye were measured to obtain the total RBF. In healthy subjects, the total RBF was 37.8 ± 6.8 µl/min. There was a significant linear correlation between retinal vessel diameter and maximal velocity (slope = 0.1016; p < 0.001; r2 = 0.8597) and a significant power curve correlation between retinal vessel diameter and blood flow (3.63 × 10-5 × D2.54; p < 0.001; r2 = 0.7287). No significant relationship was found between total RBF and systolic and diastolic blood pressure, ocular perfusion pressure, heart rate, or hematocrit. For RVO patients (n = 6), a significant decrease in RBF was noted in occluded veins (3.51 ± 2.25 µl/min) compared with the contralateral healthy eye (11.07 ± 4.53 µl/min). For occluded vessels, the slope between diameter and velocity was 0.0195 (p < 0.001; r2 = 0.6068) and the relation between diameter and flow was Q = 9.91 × 10-6 × D2.41 (p < 0.01; r2 = 0.2526). CONCLUSION: This AO-LDV prototype offers new opportunity to study RBF in humans and to evaluate treatment in retinal vein diseases.

Chemically induced cone degeneration in the 13-lined ground squirrel.

Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the in vivo cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.

Macular dystrophy in Kabuki syndrome due to de novo KMT2D variants: refining the phenotype with multimodal imaging and follow-up over 10 years: insight into pathophysiology.

BACKGROUND: Kabuki Syndrome is a rare and genetically heterogenous condition with both ophthalmic and systemic complications and typical facial features. We detail the macular phenotype in two unrelated patients with Kabuki syndrome due to de novo nonsense variants in KMT2D, one novel. A follow-up of 10 years is reported. Pathogenicity of both de novo nonsense variants is analyzed. METHODS: Four eyes of two young patients were studied by full clinical examination, kinetic perimetry, short wavelength autofluorescence, full field (ff) ERGs, and spectral-domain optical coherence tomography (SD-OCT). One patient had adaptive optic (AO) imaging. Whole exome sequencing was performed in both patients. RESULTS: Both patients had de novo nonsense variants in KMTD2. One patient had c.14843C>G; p. (Ser4948ter) novel variant and the second c.11119C>T; p. (Arg3707ter). Both had a stable Snellen visual acuity of 0.2-0.3. The retinal multimodal imaging demonstrated abnormalities at the fovea in both eyes: hyperreflectivity to blue light and a well-delimited gap-disruption of ellipsoid and interdigitation layer on OCT. The dark area on AO imaging is presumed to be absent for, or with structural change to photoreceptors. The ff ERGs and kinetic visual fields were normal. The foveal findings remained stable over several years. CONCLUSION: Kabuki syndrome-related maculopathy is a distinct loss of photoreceptors at the fovea as shown by multimodal imaging including, for the first time, AO imaging. This report adds to the literature of only one case with maculopathy with two additional macular dystrophies in patients with Kabuki syndrome. Although underestimated, these cases further raise awareness of the potential impact of retinal manifestations of Kabuki syndrome not only among ophthalmologists but also other healthcare professionals involved in the care of patients with this multisystem disorder.

Refractive extended depth-of-focus lens design based on periodic power profile for presbyopia correction.

PURPOSE: Limitations of existing diffractive multifocal designs for presbyopia correction include discrete foci and photic phenomena such as halos and glare. This study aimed to explore a methodology for developing refractive extended depth-of-focus (EDoF) lenses based on a periodic power profile. METHODS: The proposed design technique employed an optical power profile that periodically alternated between far, intermediate and near distances across the pupil radius. To evaluate the lens designs, optical bench testing was conducted. The impact on visual performance was assessed using a spatial light modulator-based adaptive optics vision simulator in human subjects. Additionally, the effects of pupil size change and lens decentration on retinal image quality were examined. A comparative performance analysis was carried out against a typical diffractive trifocal design and a monofocal lens. RESULTS: The proposed design method was found to be effective in uniformly distributing light energy across all object distances within the desired depth of focus (DoF). While trade-offs between overall image quality and DoF still exist, the EDoF lens design, when tested in human subjects, provided a continuous DoF spanning over 2.25 D. The results also revealed that the EDoF design had a slightly higher dependence on changes in pupil size and lens decentration than the diffractive trifocal design. CONCLUSION: The proposed design method showed significant potential as an approach for developing refractive EDoF ophthalmic lenses. These lenses offer a continuous DoF but are slightly more susceptible to variations in pupil size and decentration compared with the diffractive trifocal design.

Cone photoreceptor dysfunction in retinitis pigmentosa revealed by optoretinography.

Retinitis pigmentosa (RP) is the most common group of inherited retinal degenerative diseases, whose most debilitating phase is cone photoreceptor death. Perimetric and electroretinographic methods are the gold standards for diagnosing and monitoring RP and assessing cone function. However, these methods lack the spatial resolution and sensitivity to assess disease progression at the level of individual photoreceptor cells, where the disease originates and whose degradation causes vision loss. High-resolution retinal imaging methods permit visualization of human cone cells in vivo but have only recently achieved sufficient sensitivity to observe their function as manifested in the cone optoretinogram. By imaging with phase-sensitive adaptive optics optical coherence tomography, we identify a biomarker in the cone optoretinogram that characterizes individual cone dysfunction by stimulating cone cells with flashes of light and measuring nanometer-scale changes in their outer segments. We find that cone optoretinographic responses decrease with increasing RP severity and that even in areas where cone density appears normal, cones can respond differently than those in controls. Unexpectedly, in the most severely diseased patches examined, we find isolated cones that respond normally. Short-wavelength-sensitive cones are found to be more vulnerable to RP than medium- and long-wavelength-sensitive cones. We find that decreases in cone response and cone outer-segment length arise earlier in RP than changes in cone density but that decreases in response and length are not necessarily correlated within single cones.

Optics and neural adaptation jointly limit human stereovision.

Stereovision is the ability to perceive fine depth variations from small differences in the two eyes' images. Using adaptive optics, we show that even minute optical aberrations that are not clinically correctable, and go unnoticed in everyday vision, can affect stereo acuity. Hence, the human binocular system is capable of using fine details that are not experienced in everyday vision. Interestingly, stereo acuity varied considerably across individuals even when they were provided identical perfect optics. We also found that individuals' stereo acuity is better when viewing with their habitual optics rather than someone else's (better) optics. Together, these findings suggest that the visual system compensates for habitual optical aberrations through neural adaptation and thereby optimizes stereovision uniquely for each individual. Thus, stereovision is limited by small optical aberrations and by neural adaptation to one's own optics.

Optical computation of a spin glass dynamics with tunable complexity.

Spin glasses (SGs) are paradigmatic models for physical, computer science, biological, and social systems. The problem of studying the dynamics for SG models is nondetermistic polynomial-time (NP) hard; that is, no algorithm solves it in polynomial time. Here we implement the optical simulation of an SG, exploiting the N segments of a wavefront-shaping device to play the role of the spin variables, combining the interference downstream of a scattering material to implement the random couplings between the spins (the [Formula: see text] matrix) and measuring the light intensity on a number P of targets to retrieve the energy of the system. By implementing a plain Metropolis algorithm, we are able to simulate the spin model dynamics, while the degree of complexity of the potential energy landscape and the region of phase diagram explored are user defined, acting on the ratio [Formula: see text] We study experimentally, numerically, and analytically this Hopfield-like system displaying a paramagnetic, ferromagnetic, and SG phase, and we demonstrate that the transition temperature [Formula: see text] to the glassy phase from the paramagnetic phase grows with α. We demonstrate the computational advantage of the optical SG where interaction terms are realized simultaneously when the independent light rays interfere on the detector's surface. This inherently parallel measurement of the energy provides a speedup with respect to purely in silico simulations scaling with N.

Realizing the Full Potential of Advanced Microscopy Approaches for Interrogating Plant-Microbe Interactions.

Microscopy has served as a fundamental tool for insight and discovery in plant-microbe interactions for centuries. From classical light and electron microscopy to corresponding specialized methods for sample preparation and cellular contrasting agents, these approaches have become routine components in the toolkit of plant and microbiology scientists alike to visualize, probe and understand the nature of host-microbe relationships. Over the last three decades, three-dimensional perspectives led by the development of electron tomography, and especially, confocal techniques continue to provide remarkable clarity and spatial detail of tissue and cellular phenomena. Confocal and electron microscopy provide novel revelations that are now commonplace in medium and large institutions. However, many other cutting-edge technologies and sample preparation workflows are relatively unexploited yet offer tremendous potential for unprecedented advancement in our understanding of the inner workings of pathogenic, beneficial, and symbiotic plant-microbe interactions. Here, we highlight key applications, benefits, and challenges of contemporary advanced imaging platforms for plant-microbe systems with special emphasis on several recently developed approaches, such as light-sheet, single molecule, super-resolution, and adaptive optics microscopy, as well as ambient and cryo-volume electron microscopy, X-ray microscopy, and cryo-electron tomography. Furthermore, the potential for complementary sample preparation methodologies, such as optical clearing, expansion microscopy, and multiplex imaging, will be reviewed. Our ultimate goal is to stimulate awareness of these powerful cutting-edge technologies and facilitate their appropriate application and adoption to solve important and unresolved biological questions in the field. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Adaptive Optics Microscopy with Wavefront Sensing Based on Neighbor Correlation.

Complex structures in living cells and tissues induce wavefront errors when light waves pass through them, and images observed with optical microscopes are undesirably blurred. This problem is especially serious for living plant cells because images are strikingly degraded even within a single cell. Adaptive optics (AO) is expected to be a solution to this problem by correcting such wavefront errors, thus enabling high-resolution imaging. In particular, scene-based AO involves wavefront sensing based on the image correlation between subapertures in a Shack-Hartmann wavefront sensor and thus does not require an intense point light source. However, the complex 3D structures of living cells often cause low correlation between subimages, leading to loss of accuracy in wavefront sensing. This paper proposes a novel method for scene-based sensing using only image correlations between adjacent subapertures. The method can minimize changes between subimages to be correlated and thus prevent inaccuracy in phase estimation. Using an artificial test target mimicking the optical properties of a layer of living plant cells, an imaging performance with a Strehl ratio of approximately 0.5 was confirmed. Upon observation of chloroplast autofluorescence inside living leaf cells of the moss Physcomitrium patens, recovered resolution images were successfully obtained even with complex biological structures. Under bright-field illumination, the proposed method outperformed the conventional method, demonstrating the future potential of this method for label- and damage-free AO microscopy. Several points for improvement in terms of the effect of AO correction are discussed.

Python-Microscope - a new open-source Python library for the control of microscopes.

Custom-built microscopes often require control of multiple hardware devices and precise hardware coordination. It is also desirable to have a solution that is scalable to complex systems and that is translatable between components from different manufacturers. Here we report Python-Microscope, a free and open-source Python library for high-performance control of arbitrarily complex and scalable custom microscope systems. Python-Microscope offers simple to use Python-based tools, abstracting differences between physical devices by providing a defined interface for different device types. Concrete implementations are provided for a range of specific hardware, and a framework exists for further expansion. Python-Microscope supports the distribution of devices over multiple computers while maintaining synchronisation via highly precise hardware triggers. We discuss the architectural features of Python-Microscope that overcome the performance problems often raised against Python and demonstrate the different use cases that drove its design: integration with user-facing projects, namely the Microscope-Cockpit project; control of complex microscopes at high speed while using the Python programming language; and use as a microscope simulation tool for software development.

Data-driven modeling and control of an X-ray bimorph adaptive mirror.

Adaptive X-ray mirrors are being adopted on high-coherent-flux synchrotron and X-ray free-electron laser beamlines where dynamic phase control and aberration compensation are necessary to preserve wavefront quality from source to sample, yet challenging to achieve. Additional difficulties arise from the inability to continuously probe the wavefront in this context, which demands methods of control that require little to no feedback. In this work, a data-driven approach to the control of adaptive X-ray optics with piezo-bimorph actuators is demonstrated. This approach approximates the non-linear system dynamics with a discrete-time model using random mirror shapes and interferometric measurements as training data. For mirrors of this type, prior states and voltage inputs affect the shape-change trajectory, and therefore must be included in the model. Without the need for assumed physical models of the mirror's behavior, the generality of the neural network structure accommodates drift, creep and hysteresis, and enables a control algorithm that achieves shape control and stability below 2 nm RMS. Using a prototype mirror and ex situ metrology, it is shown that the accuracy of our trained model enables open-loop shape control across a diverse set of states and that the control algorithm achieves shape error magnitudes that fall within diffraction-limited performance.

Structural and functional analysis of retinal vasculature in HANAC syndrome with a novel intronic COL4A1 mutation.

PURPOSE: Mutations of the COL4A1 gene, a major structural protein of vessels, may cause hereditary angiopathy with nephropathy, aneurysms and muscle cramps (HANAC) syndrome. The vascular structure and function of patients with HANAC is poorly known. Here, we report a family with HANAC syndrome associated to a previously unreported mutation in COL4A1. The structure and function of retinal vessels were detailed by adaptive optics ophthalmoscopy (AOO) and optical coherence tomography (OCT) angiography. METHODS: Clinical data from six affected individuals (43 to 72 years old) from a single family comprising two generations were collected. Imaging charts including conventional fundus imaging, OCT-angiography and AOO in static and dynamic (flicker) mode were reviewed. DNA sequencing was done in the proband. RESULTS: DNA sequencing of the proband revealed a heterozygous deletion of COL4A1 (NM_001845) at position 1120 in the intron 20 resulting in the loss of splicing donor site for exon 20 (c.1120 + 2_1120 + 8del heterozygote). Four patients had arterial hypertension, and three had kidney dysfunction, one of which under dialysis. By fundus examination, five had typical retinal arteriolar tortuosity with arteriolar loops. Wall-to-lumen ratio of arteries was within normal limits, that is, lower than expected for hypertensive patients. Several foci of arteriolar irregularities were noted in the two oldest patients. In three affected subjects, evaluation of the neurovascular coupling showed a higher flicker-induced vasodilation than a control population (6 % to 11 %; n < 5 %). CONCLUSIONS: Structural and dynamic analysis of retinal vessels in a HANAC family bearing a previously unreported intronic COL4 mutation was done. In addition to arteriolar tortuosity, we found reduced wall-to-lumen ratio, arteriolar irregularity and increased vasodilatory response to flicker light. These abnormalities were more marked in the oldest subjects. This abnormal flicker response affected also non-tortuous arteries, suggesting that microvascular dysfunction extends beyond tortuosity. Such explorations may help to better vascular dysfunction related to HANAC and hence better understand the mechanisms of end-organ damage.

In-depth polarisation resolved SHG microscopy in biological tissues using iterative wavefront optimisation.

Polarised nonlinear microscopy has been extensively developed to study molecular organisation in biological tissues, quantifying the response of nonlinear signals to a varying incident linear polarisation. Polarisation Second harmonic Generation (PSHG) in particular is a powerful tool to decipher sub-microscopic modifications of fibrillar collagen organisation in type I and III collagen-rich tissues. The quality of SHG imaging is however limited to about one scattering mean free path in depth (typically 100 micrometres in biological tissues), due to the loss of focus quality, induced by wavefront aberrations and scattering at even larger depths. In this work, we study how optical depth penetration in biological tissues affects the quality of polarisation control, a crucial parameter for quantitative assessment of PSHG measurements. We apply wavefront shaping to correct for SHG signal quality in two regimes, adaptive optics for smooth aberration modes corrections at shallow depth, and wavefront shaping of higher spatial frequencies for optical focus correction at larger depths. Using nonlinear SHG active nanocrystals as guide stars, we quantify the capabilities of such optimisation methods to recover a high-quality linear polarisation and investigate how this approach can be applied to in-depth PSHG imaging in tissues, namely tendon and mouse cranial bone.