Model-based correction of rapid thermal confounds in fluorescence neuroimaging of targeted perturbation.

Significance: An array of techniques for targeted neuromodulation is emerging, with high potential in brain research and therapy. Calcium imaging or other forms of functional fluorescence imaging are central solutions for monitoring cortical neural responses to targeted neuromodulation, but often are confounded by thermal effects that are inter-mixed with neural responses. Aim: Here, we develop and demonstrate a method for effectively suppressing fluorescent thermal transients from calcium responses. Approach: We use high precision phased-array 3 MHz focused ultrasound delivery integrated with fiberscope-based widefield fluorescence to monitor cortex-wide calcium changes. Our approach for detecting the neural activation first takes advantage of the high inter-hemispheric correlation of resting state Ca2+ dynamics and then removes the ultrasound-induced thermal effect by subtracting its simulated spatio-temporal signature from the processed profile. Results: The focused 350 µm-sized ultrasound stimulus triggered rapid localized activation events dominated by transient thermal responses produced by ultrasound. By employing bioheat equation to model the ultrasound heat deposition, we can recover putative neural responses to ultrasound. Conclusions: The developed method for canceling transient thermal fluorescence quenching could also find applications with optical stimulation techniques to monitor thermal effects and disentangle them from neural responses. This approach may help deepen our understanding of the mechanisms and macroscopic effects of ultrasound neuromodulation, further paving the way for tailoring the stimulation regimes toward specific applications.

Shedding light on ultrasound in action: Optical and optoacoustic monitoring of ultrasound brain interventions.

Monitoring brain responses to ultrasonic interventions is becoming an important pillar of a growing number of applications employing acoustic waves to actuate and cure the brain. Optical interrogation of living tissues provides a unique means for retrieving functional and molecular information related to brain activity and disease-specific biomarkers. The hybrid optoacoustic imaging methods have further enabled deep-tissue imaging with optical contrast at high spatial and temporal resolution. The marriage between light and sound thus brings together the highly complementary advantages of both modalities toward high precision interrogation, stimulation, and therapy of the brain with strong impact in the fields of ultrasound neuromodulation, gene and drug delivery, or noninvasive treatments of neurological and neurodegenerative disorders. In this review, we elaborate on current advances in optical and optoacoustic monitoring of ultrasound interventions. We describe the main principles and mechanisms underlying each method before diving into the corresponding biomedical applications. We identify areas of improvement as well as promising approaches with clinical translation potential.

Enhancing optoacoustic mesoscopy through calibration-based iterative reconstruction.

Optoacoustic mesoscopy combines rich optical absorption contrast with high spatial resolution at tissue depths beyond reach for microscopic techniques employing focused light excitation. The mesoscopic imaging performance is commonly hindered by the use of inaccurate delay-and-sum reconstruction approaches and idealized modeling assumptions. In principle, image reconstruction performance could be enhanced by simulating the optoacoustic signal generation, propagation, and detection path. However, for most realistic experimental scenarios, the underlying total impulse response (TIR) cannot be accurately modelled. Here we propose to capture the TIR by scanning of a sub-resolution sized absorber. Significant improvement of spatial resolution and depth uniformity is demonstrated over 3 mm range, outperforming delay-and-sum and model-based reconstruction implementations. Reconstruction performance is validated by imaging subcutaneous murine vasculature and human skin in vivo. The proposed experimental calibration and reconstruction paradigm facilitates quantitative inversions while averting complex physics-based simulations. It can readily be applied to other imaging modalities employing TIR-based reconstructions.

Guided Waves in the Skull.

Skull bone is the main obstacle for transcranial ultrasound therapy and imaging applications. Most efforts in characterizing ultrasonic properties of the skull have been limited to a narrow frequency range and normal incidence. On the other hand, acoustic guided waves in plates have been used in non-destructive evaluation of materials and also to assess the strength of long bones. Recent work has likewise revealed the existence of skull-guided waves (SGWs) in mice and humans when performing measurements over a broad range of frequencies and incidence angles. Here we provide an overview on the recent progress in our understanding on the propagation of SGWs, describe the measurement techniques used to detect SGWs, the experimental observations, and the accompanying modeling efforts. Finally, the outstanding challenges to harness SGWs in applications such as transcranial therapy, imaging, and cranial bone assessment are discussed.

Tracking Strain-Specific Morphogenesis and Angiogenesis of Murine Calvaria with Large-Scale Optoacoustic and Ultrasound Microscopy.

Skull bone development is a dynamic and well-coordinated process playing a key role in maturation and maintenance of the bone marrow (BM), fracture healing, and progression of diseases such as osteoarthritis or osteoporosis. At present, dynamic transformation of the growing bone (osteogenesis) as well as its vascularization (angiogenesis) remain largely unexplored due to the lack of suitable in vivo imaging techniques capable of noninvasive visualization of the whole developing calvaria at capillary-level resolution. We present a longitudinal study on skull bone development using ultrasound-aided large-scale optoacoustic microscopy (U-LSOM). Skull bone morphogenesis and microvascular growth patterns were monitored in three common mouse strains (C57BL/6J, CD-1, and Athymic Nude-Foxn1nu) at the whole-calvaria scale over a 3-month period. Strain-specific differences in skull development were revealed by quantitative analysis of bone and vessel parameters, indicating the coupling between angiogenesis and osteogenesis during skull bone growth in a minimally invasive and label-free manner. The method further enabled identifying BM-specific sinusoidal vessels, and superficial skull vessels penetrating into BM compartments. Our approach furnishes a new high-throughput longitudinal in vivo imaging platform to study morphological and vascular skull alterations in health and disease, shedding light on the critical links between blood vessel formation, skull growth, and regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

High-resolution fluorescence-guided transcranial ultrasound mapping in the live mouse brain.

Understanding the physiological impact of transcranial ultrasound in rodent brains may offer an important preclinical model for human scale magnetic resonance­guided focused ultrasound methods. However, precision tools for high-resolution transcranial ultrasound targeting and real-time in vivo tracking of its effects at the mouse brain scale are currently lacking. We report a versatile bidirectional hybrid fluorescence-ultrasound (FLUS) system incorporating a 0.35-mm precision spherical-phased array ultrasound emission with a fiberscope-based wide-field fluorescence imaging. We show how the marriage between cortex-wide functional imaging and targeted ultrasound delivery can be used to transcranially map previously undocumented localized fluorescence events caused by reversible thermal processes and perform high-speed large-scale recording of neural activity induced by focused ultrasound. FLUS thus naturally harnesses the extensive toolbox of fluorescent tags and ultrasound's localized bioeffects toward visualizing and causally perturbing a plethora of normal and pathophysiological processes in the living murine brain.

Spherical Array System for High-Precision Transcranial Ultrasound Stimulation and Optoacoustic Imaging in Rodents.

Ultrasound can be delivered transcranially to ablate brain tissue, open the blood-brain barrier, or affect neural activity. Transcranial focused ultrasound in small rodents is typically done with low-frequency single-element transducers, which results in unspecific targeting and impedes the concurrent use of fast neuroimaging methods. In this article, we devised a wide-angle spherical array bidirectional interface for high-resolution parallelized optoacoustic imaging and transcranial ultrasound (POTUS) delivery in the same target regions. The system operates between 3 and 9 MHz, allowing to generate and steer focal spots with widths down to [Formula: see text] across a field of view covering the entire mouse brain, while the same array is used to capture high-resolution 3-D optoacoustic data in real time. We showcase the system's versatile beam-forming capacities as well as volumetric optoacoustic imaging capabilities and discuss its potential to noninvasively monitor brain activity and various effects of ultrasound emission.

Mental Health Problems among COVID-19 Frontline Healthcare Workers and the Other Country-Level Epidemics: The Case of Mexico.

COVID-19 frontline healthcare workers (FHCW) are struggling to cope with challenges that threaten their wellbeing. We examine the frequency and predictors of the most frequent mental health problems (MHP) among FHCW during the first COVID-19 peak in Mexico, one of the most severely affected countries in terms of FHCW's COVID-19 mortality. A cross-sectional survey was conducted between May 8 and August 18, 2020. A total of 47.5% of the sample (n = 2218) were FHCW. The most frequent MHP were insomnia, depression, posttraumatic stress symptoms, and health anxiety/somatization (whole sample: 45.7, 37.4, 33.9, and 21.3%; FHCW: 52.4, 43.4, 40.3 and 26.1, respectively). As compared to during the initial COVID-19 phase, depression and health anxiety/somatization symptoms as well as experiences of grieving due to COVID-19, personal COVID-19 status, and having relatives and close friends with COVID-19 were more frequent during the COVID-19 peak. Obesity, domestic violence, personal COVID-19 status, and grieving because of COVID-19 were included in regression models for main FHCW's MHP during the COVID-19 peak. In conclusion, measures to decrease other country-level epidemics contributing to the likelihood of COVID-19 complications (obesity) and MHP (domestic violence) as well as FHCW´s probability of COVID-19 infection could safeguard not only their physical but also mental health.

Rapid functional optoacoustic micro-angiography in a burst mode.

Optoacoustic microscopy (OAM) can image intrinsic optical absorption contrast at depths of several millimeters where state-of-the-art optical microscopy techniques fail due to intense light scattering in living tissues. Yet, wide adoption of OAM in biology and medicine is hindered by slow image acquisition speed, small field of view (FOV), and/or lack of spectral differentiation capacity of common system implementations. We report on a rapid acquisition functional optoacoustic micro-angiography approach that employs a burst-mode laser triggering scheme to simultaneously acquire multi-wavelength 3D images over an extended FOV covering ${50}\;{\rm mm} \times {50}\;{\rm mm}$50mm×50mm in a single mechanical overfly scan, attaining 28 µm and 14 µm resolution in lateral and axial dimensions, respectively. Owing to an ultrawideband low-noise design featuring a spherically focused polyvinylidene difluoride transducer, we demonstrate imaging of human skin and underlying vasculature at up to 3.8 mm depth when using per-pulse laser energies of only 25 µJ without employing signal averaging. Overall, the developed system greatly enhances performance and usability of OAM for dermatologic and micro-angiographic studies.

Discerning calvarian microvascular networks by combined optoacoustic ultrasound microscopy.

Bone microvasculature plays a paramount role in bone marrow maintenance, development, and hematopoiesis. Studies of calvarian vascular patterns within living mammalian skull with the available intravital microscopy techniques are limited to small scale observations. We developed an optical-resolution optoacoustic microscopy method combined with ultrasound biomicroscopy in order to reveal and discern the intricate networks of calvarian and cerebral vasculature over large fields of view covering majority of the murine calvaria. The vasculature segmentation method is based on an angle-corrected homogeneous model of the rodent skull, generated using simultaneously acquired three-dimensional pulse-echo ultrasound images. The hybrid microscopy design along with the appropriate skull segmentation method enable high throughput studies of a living bone while facilitating correct anatomical interpretation of the vasculature images acquired with optical resolution optoacoustic microscopy.

Intravital optoacoustic and ultrasound bio-microscopy reveal radiation-inhibited skull angiogenesis.

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators.

Functional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs.

Observation of Guided Acoustic Waves in a Human Skull.

Human skull poses a significant barrier for the propagation of ultrasound waves. Development of methods enabling more efficient ultrasound transmission into and from the brain is therefore critical for the advancement of ultrasound-mediated transcranial imaging or actuation techniques. We report on the first observation of guided acoustic waves in the near field of an ex vivo human skull specimen in the frequency range between 0.2 and 1.5MHz. In contrast to what was previously observed for guided wave propagation in thin rodent skulls, the guided wave observed in a higher-frequency regime corresponds to a quasi-Rayleigh wave, confined mostly to the cortical bone layer. The newly discovered near-field properties of the human skull are expected to facilitate the development of more efficient diagnostic and therapeutic techniques based on transcranial ultrasound.

Dual-wavelength hybrid optoacoustic-ultrasound biomicroscopy for functional imaging of large-scale cerebral vascular networks.

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade-offs between the spatiotemporal resolution, field-of-view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual-wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large-scale cerebral vascular networks. The system offers 3-dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 × 8 mm2 , thus covering the entire cortical vasculature in mice. The large-scale OA imaging capacity is complemented by simultaneously acquired pulse-echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.

Virtual craniotomy for high-resolution optoacoustic brain microscopy.

Ultrasound-mediated transcranial images of the brain often suffer from acoustic distortions produced by the skull bone. In high-resolution optoacoustic microscopy, the skull-induced acoustic aberrations are known to impair image resolution and contrast, further skewing the location and intensity of the different absorbing structures. We present a virtual craniotomy deconvolution algorithm based on an ultrasound wave propagation model that corrects for the skull-induced distortions in optically-resolved optoacoustic transcranial microscopy data. The method takes advantage of the geometrical and spectral information of a pulse-echo ultrasound image of the skull simultaneously acquired by our multimodal imaging system. Transcranial mouse brain imaging experiments confirmed the ability to accurately account for the signal amplitude decay, temporal delay and pulse broadening introduced by the rodent's skull. Our study is the first to demonstrate skull-corrected transcranial optoacoustic imaging in vivo.

Prediction and near-field observation of skull-guided acoustic waves.

Ultrasound waves propagating in water or soft biological tissue are strongly reflected when encountering the skull, which limits the use of ultrasound-based techniques in transcranial imaging and therapeutic applications. Current knowledge on the acoustic properties of the cranial bone is restricted to far-field observations, leaving its near-field unexplored. We report on the existence of skull-guided acoustic waves, which was herein confirmed by near-field measurements of optoacoustically-induced responses in ex-vivo murine skulls immersed in water. Dispersion of the guided waves was found to reasonably agree with the prediction of a multilayered flat plate model. We observed a skull-guided wave propagation over a lateral distance of at least 3 mm, with a half-decay length in the direction perpendicular to the skull ranging from 35 to 300 µm at 6 and 0.5 MHz, respectively. Propagation losses are mostly attributed to the heterogenous acoustic properties of the skull. It is generally anticipated that our findings may facilitate and broaden the application of ultrasound-mediated techniques in brain diagnostics and therapy.

Broadband acoustic properties of a murine skull.

It has been well recognized that the presence of a skull imposes harsh restrictions on the use of ultrasound and optoacoustic techniques in the study, treatment and modulation of the brain function. We propose a rigorous modeling and experimental methodology for estimating the insertion loss and the elastic constants of the skull over a wide range of frequencies and incidence angles. A point-source-like excitation of ultrawideband acoustic radiation was induced via the absorption of nanosecond duration laser pulses by a 20 µm diameter microsphere. The acoustic waves transmitted through the skull are recorded by a broadband, spherically focused ultrasound transducer. A coregistered pulse-echo ultrasound scan is subsequently performed to provide accurate skull geometry to be fed into an acoustic transmission model represented in an angular spectrum domain. The modeling predictions were validated by measurements taken from a glass cover-slip and ex vivo adult mouse skulls. The flexible semi-analytical formulation of the model allows for seamless extension to other transducer geometries and diverse experimental scenarios involving broadband acoustic transmission through locally flat solid structures. It is anticipated that accurate quantification and modeling of the skull transmission effects would ultimately allow for skull aberration correction in a broad variety of applications employing transcranial detection or transmission of high frequency ultrasound.

Effects of the murine skull in optoacoustic brain microscopy.

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

Influence of the absorber dimensions on wavefront shaping based on volumetric optoacoustic feedback.

The recently demonstrated control over light distribution through turbid media based on real-time three-dimensional optoacoustic feedback has offered promising prospects to interferometrically focus light within scattering objects. Nevertheless, the focusing capacity of the feedback-based approach is strongly conditioned by the number of optical modes (speckle grains) enclosed in the volume that can be resolved with the optoacoustic imaging system. In this Letter, we experimentally tested the light intensity enhancement achieved with optoacoustic feedback measurements from different sizes of absorbing microparticles. The importance of the obtained results is discussed in the context of potential signal enhancement at deep locations within a scattering medium where the effective speckle grain sizes approach the minimum values dictated by optical diffraction.

Volumetric tracking of migratory melanophores during zebrafish development by optoacoustic microscopy.

Unveiling mechanisms driving specification, recruitment and regeneration of melanophores is key in understanding melanin-related disorders. This study reports on the applicability of a hybrid focus optoacoustic microscope (HFOAM) for volumetric tracking of migratory melanophores in developing zebrafish. The excellent contrast from highly-absorbing melanin provided by the method is shown to be ideal for label-free dynamic visualization of melanophores in their unperturbed environment. We established safe laser energy levels that enable high-contrast longitudinal tracking of the cells over an extended period of developmental time without causing cell toxicity or pigment bleaching. Owing to its hybrid optical and acoustic resolution, the new imaging technique can be seamlessly applied for noninvasive studies of both optically-transparent larval as well as adult stages of the zebrafish model organism, which is not possible using other optical microscopy methods.