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Optogenetics employs engineered viruses to genetically modify cells to express specific light-sensitive ion channels. The standard method for gene delivery in the brain involves invasive craniotomies that expose the brain and direct injections of viruses that invariably damage neural tissue along the syringe tract. A recently proposed alternative in which non-invasive optogenetics is performed with focused ultrasound (FUS)-mediated blood-brain barrier (BBB) openings has been found to non-invasively facilitate gene delivery for optogenetics in mice. Although gene delivery can be performed non-invasively, validating successful viral transduction and expression of encoded ion channels in target tissue typically involves similar invasive techniques, such as craniotomies in longitudinal studies and/or postmortem histology. Functional ultrasound imaging (fUSi) is an emerging neuroimaging technique that can be used to transcranially detect changes in cerebral blood volume following introduction of a stimulus. In this study, we implemented a fully non-invasive combined FUS-fUSi technique for performing optogenetics in mice. FUS successfully delivered viruses encoding the red-shifted channelrhodopsin variant ChrimsonR in all treated subjects. fUSi successfully identified stimulus-evoked cerebral blood volume changes preferentially in brain regions expressing the light-sensitive ion channels. Improvements in cell-specific targeting of viral vectors and transcranial ultrasound imaging will make the combined technique a useful tool for neuroscience research in small animals.

Conventional biventricular (BiV) pacing cardiac resynchronization therapy (CRT) is an established treatment for heart failure patients. Recently, multiple novel CRT delivering technologies such as His-Bundle pacing have been investigated as alternative pacing strategies for optimal treatment benefit. Electromechanical Wave Imaging (EWI), a high frame-rate echocardiography-based modality, is capable of visualizing the change from dyssynchronous activation to resynchronized BiV-paced ventricles in 3D. This proof-of-concept study introduces a new EWI-based dispersion metric to further characterize ventricular activation. Patients with His-Bundle device implantation (n=4), left-bundle branch block (n=10), right-ventricular (RV) pacing (n=10), or BiV pacing (n=15) were imaged, as well as four volunteers in normal sinus rhythm (NSR). EWI successfully mapped the ventricular activation resulting from His-Bundle pacing. Additionally, very similar activation patterns were obtained in the NSR subjects, confirming recovery of physiological activation with His pacing. The dispersion metric was the most sensitive EWI-based metric that identified His pacing as the most efficient treatment (lowest activation time spread), followed by BiV and RV pacing. More specifically, the dispersion metric significantly (p

Mapping of mechanical properties, dependent on the frequency of motion, is relevant in diagnosis, monitoring treatment response, or intra-operative surgical resection planning. While shear wave speeds at different frequencies have been described elsewhere, the effect of frequency on the "on-axis" acoustic radiation force (ARF)-induced displacement has not been previously investigated. Instead of generating single transducer-harmonic motion imaging (ST-HMI)-derived peak-to-peak displacement (P2PD) image at a particular frequency, a novel multi-frequency excitation pulse is proposed to generate P2PD images at 100-1000 Hz simultaneously. The performance of the proposed excitation pulse is compared with the ARFI by imaging 16 different inclusions (Young's moduli of 6, 9, 36, 70 kPa and diameters of 1.6, 2.5, 6.5, and 10.4 mm) embedded in an 18 kPa background. Depending on inclusion size and stiffness, the maximum CNR and contrast were achieved at different frequencies and were always higher than ARFI. The frequency, at which maximum CNR and contrast were achieved, increased with stiffness for fixed inclusion's size and decreased with size for fixed stiffness. In vivo feasibility is tested by imaging a 4T1 breast cancer mouse tumor on Day 6, 12, and 19 post-injection of tumor cells. Similar to phantoms, the CNR of ST-HMI images was higher than ARFI and increased with frequency for the tumor on Day 6. Besides, P2PD at 100-1000 Hz indicated that the tumor became stiffer with respect to the neighboring non-cancerous tissue over time. These results indicate the importance of using a multi-frequency excitation pulse to simultaneously generate displacement at multiple frequencies to better delineate inclusions or tumors.

Non-invasive monitoring of atherosclerosis remains challenging. Pulse Wave Imaging (PWI) is a non-invasive technique to measure the local stiffness at diastolic and end-systolic pressures and quantify the hemodynamics. The objective of this study is twofold, namely (1) to investigate the capability of (adaptive) PWI to assess progressive change in local stiffness and homogeneity of the carotid in a high-cholesterol swine model and (2) to assess the ability of PWI to monitor the change in hemodynamics and a corresponding change in stiffness. Nine (n=9) hypercholesterolemic swine were included in this study and followed for up to 9 months. A ligation in the left carotid was used to cause a hemodynamic disturbance. The carotids with detectable hemodynamic disturbance showed a reduction in wall shear stress immediately after ligation (2.12 ± 0.49 to 0.98 ± 0.47 Pa for 40–90% ligation (Group B) and 1.82 ± 0.25 to 0.49 ± 0.46 Pa for >90% ligation (Group C)). Histology revealed subsequent lesion formation after 8–9 months, and the type of lesion formation was dependent on the type of the induced ligation, with more complex plaques observed in the carotids with a more significant ligation (C: >90%). The compliance progression appears differed for groups B and C, with an increase in compliance to 2.09 ± 2.90×10−10 m2 Pa−1 for group C whereas the compliance of group B remained low at 8 months (0.95 ± 0.94×10−10 m2 Pa−1). In summary, PWI appeared capable of monitoring a change in wall shear stress and separating two distinct progression pathways resulting in distinct compliances.

Purpose:Pancreatic ductal adenocarcinoma (PDA) is a common, deadly cancer that is challenging both to diagnose and to manage. Its hallmark is an expansive, desmoplastic stroma characterized by high mechanical stiffness. In this study, we sought to leverage this feature of PDA for two purposes: differential diagnosis and monitoring of response to treatment.Experimental Design:Harmonic motion imaging (HMI) is a functional ultrasound technique that yields a quantitative relative measurement of stiffness suitable for comparisons between individuals and over time. We used HMI to quantify pancreatic stiffness in mouse models of pancreatitis and PDA as well as in a series of freshly resected human pancreatic cancer specimens.Results:In mice, we learned that stiffness increased during progression from preneoplasia to adenocarcinoma and also effectively distinguished PDA from several forms of pancreatitis. In human specimens, the distinction of tumors versus adjacent pancreatitis or normal pancreas tissue was even more stark. Moreover, in both mice and humans, stiffness increased in proportion to tumor size, indicating that tuning of mechanical stiffness is an ongoing process during tumor progression. Finally, using a brca2–mutant mouse model of PDA that is sensitive to cisplatin, we found that tissue stiffness decreases when tumors respond successfully to chemotherapy. Consistent with this observation, we found that tumor tissues from patients who had undergone neoadjuvant therapy were less stiff than those of untreated patients.Conclusions:These findings support further development of HMI for clinical applications in disease staging and treatment response assessment in PDA.

Supplementary Methods

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Imaging applications tailored towards ultrasound-based treatment, such as high intensity focused ultrasound (FUS), where higher power ultrasound generates a radiation force for ultrasound elasticity imaging or therapeutics/theranostics, are affected by interference from FUS. The artifact becomes more pronounced with intensity and power. To overcome this limitation, we propose FUS-net, a method that incorporates a CNN-based U-net autoencoder trained end-to-end on 'clean' and 'corrupted' RF data in Tensorflow 2.3 for FUS artifact removal. The network learns the representation of RF data and FUS artifacts in latent space so that the output of corrupted RF input is clean RF data. We find that FUS-net perform 15% better than stacked autoencoders (SAE) on evaluated test datasets. B-mode images beamformed from FUS-net RF shows superior speckle quality and better contrast-to-noise (CNR) than both notch-filtered and adaptive least means squares filtered RF data. Furthermore, FUS-net filtered images had lower errors and higher similarity to clean images collected from unseen scans at all pressure levels. Lastly, FUS-net RF can be used with existing cross-correlation speckle-tracking algorithms to generate displacement maps. FUS-net currently outperforms conventional filtering and SAEs for removing high pressure FUS interference from RF data, and hence may be applicable to all FUS-based imaging and therapeutic methods.

Noninvasive brain stimulation using focused ultrasound (FUS) has many potential applications as a research and clinical tool, including incorporation into neural prosthetics for cognitive rehabilitation. To develop this technology, it is necessary to evaluate the safety and efficacy of FUS neuromodulation for specific brain targets and cognitive functions. It is also important to test whether repeated long-term application of FUS to deep brain targets improves or degrades behavioral and cognitive function. To this end, we investigated the effects of FUS in the dorsal striatum of nonhuman primates (NHP) performing a visual-motor decision-making task for small or large rewards. Over the course of 2 years, we performed 129 and 147 FUS applications, respectively, in two NHP. FUS (0.5 MHz @ 0.2 – 0.8 MPa) was applied to the putamen and caudate in both hemispheres to evaluate the effects on movement accuracy, motivation, decision accuracy, and response time. Sonicating the caudate or the putamen unilaterally resulted in modest but statistically significant improvements in motivation and decision accuracy, but at the cost of slower reaction times. The effects were dose (i.e., FUS pressure) and reward dependent. There was no effect on reaching accuracy, nor was there long-term behavioral impairment or neurological trauma evident on T1-weighted, T2-weighted, or susceptibility-weighted MRI scans. Sonication also resulted in significant changes in resting state functional connectivity between the caudate and multiple cortical regions. The results indicate that applying FUS to the dorsal striatum can positively impact the motivational and cognitive aspects of decision making. The capability of FUS to improve motivation and cognition in NHPs points to its therapeutic potential in treating a wide variety of human neural diseases, and warrants further development as a novel technique for non-invasive deep brain stimulation.

Focused ultrasound (FUS) in combination with systemically injected microbubbles can be used to non-invasively open the blood-brain barrier (BBB) in targeted regions for a variety of therapeutic applications. Over the past two decades, preclinical research into the safety and efficacy of FUS-induced BBB opening has proven this technique to be transient and efficacious, propelling FUS-induced BBB opening into several clinical trials in recent years. However, as clinical trials further progress, the neuroinflammatory response to FUS-induced BBB opening needs to be better understood. In this study, we provide further insight into the relationship of microbubble cavitation and the resulting innate immune response to FUS-induced BBB opening. By keeping ultrasound parameters fixed (i.e. frequency, pressure, pulse length, etc.), three groups of mice were sonicated using a real-time cavitation controller until a target cavitation dose was reached (1 [Formula: see text] 10(7) V(2)•s, 5 [Formula: see text] 10(7) V(2)•s, 1 [Formula: see text] 10(8) V(2)•s). The change in relative gene expression of the mouse inflammatory cytokines and receptors were evaluated at three different time-points (6 h, 24 h, and 72 h) after FUS. At both 6 and 24 h time-points, significant changes in relative gene expression of inflammatory cytokines and receptors were observed across all cavitation groups. However, the degree of changes in relative expression levels and the number of genes with significant changes in expression varied across the cavitation groups. Groups with a higher cavitation dose exhibited both greater changes in relative expression levels and greater number of significant changes. By 72 h, the gene expression levels returned to baseline in all cavitation dose groups, signifying a transient inflammatory response to FUS-induced BBB opening at the targeted cavitation dose levels. Furthermore, the real-time cavitation controller was able to produce consistent and significantly different BBB permeability enhancement volumes across the three different cavitation dose groups. These results indicate that cavitation monitoring and controlling during FUS-induced BBB opening can be used to potentially modulate or limit the degree of neuroinflammation, further emphasizing the importance of implementing cavitation controllers as FUS-induced BBB opening is translated into the clinic.

Harmonic motion imaging (HMI) is an ultrasound-based elasticity imaging technique that utilizes oscillatory acoustic radiation force to estimate the mechanical properties of tissues, as well as monitor high-intensity focused ultrasound (HIFU) treatment. Conventionally, in HMI, a focused ultrasound (FUS) transducer generates oscillatory tissue displacements, and an imaging transducer acquires channel data for displacement estimation, with each transducer being driven with a separate system. The fixed position of the FUS focal spot requires mechanical translation of the transducers, which can be a time-consuming and challenging procedure. In this study, we develop and characterize a new HMI system with a multi-element FUS transducer with the capability of electronic focal steering of ±5 mm and ±2 mm from the geometric focus in the axial and lateral directions, respectively. A pulse sequence was developed to drive both the FUS and imaging transducers using a single ultrasound data acquisition (DAQ) system. The setup was validated on a tissue-mimicking phantom with embedded inclusions. Integrating beam steering with the mechanical translation of the transducers resulted in a consistent high contrast to noise ratio (CNR) for the inclusions with Young's moduli of 22 and 44 kPa within a 5-kPa background while the data acquisition speed is increased by 4.5-5.2-fold compared to the case when only mechanical movements were applied. The feasibility of simultaneous generation of multiple foci and tracking the induced displacements is demonstrated in phantoms for applications where imaging or treatment of a larger region is needed. Moreover, preliminary feasibility is shown in a human subject with a breast tumor, where the mean HMI displacement within the tumor was about 4 times lower than that within perilesional tissues. The proposed HMI system facilitates data acquisition in terms of flexibility and speed and can be potentially used in the clinic for breast cancer imaging and treatment.

Objective: Focused ultrasound (FUS) has emerged as a non-invasive technique to locally and reversibly disrupt the blood-brain barrier (BBB). Here, we investigate the use of diffusion tensor imaging (DTI) as a means of detecting FUS-induced BBB opening at the absence of an MRI contrast agent. A non-human primate (NHP) was repeatedly treated with FUS and preformed circulating microbubbles to transiently disrupt the BBB (n = 4). T1- and diffusion-weighted MRI scans were acquired after the ultrasound treatment, with and without gadolinium-based contrast agent, respectively. Both scans were registered with a high-resolution T1-weighted scan of the NHP to investigate signal correlations. DTI detected an increase in fractional anisotropy from 0.21 ± 0.02 to 0.38 ± 0.03 (82.6 ± 5.2% change) within the targeted area one hour after BBB opening. Enhanced DTI contrast overlapped by 77.22 ± 9.2% with hyper-intense areas of gadolinium-enhanced T1-weighted scans, indicating diffusion anisotropy enhancement only within the BBB opening volume. Diffusion was highly anisotropic and unidirectional within the treated brain region, as indicated by the direction of the principal diffusion eigenvectors. Polar and azimuthal angle ranges decreased by 35.6% and 82.4%, respectively, following BBB opening. Evaluation of the detection methodology on a second NHP (n = 1) confirmed the across-animal feasibility of the technique. In conclusion, DTI may be used as a contrast-free MR imaging modality in lieu of contrast-enhanced T1 mapping for detecting BBB opening during focused-ultrasound treatment or evaluating BBB integrity in brain-related pathologies.

WSS measurement is challenging since it requires sensitive flow measurements at a distance close to the wall. The aim of this study is to develop an ultrasound imaging technique which combines vector flow imaging with an unsupervised data clustering approach that automatically detects the region close to the wall with optimally linear flow profile, to provide direct and robust WSS estimation. The proposed technique was evaluated in phantoms, mimicking normal and atherosclerotic vessels, and spatially registered Fluid Structure Interaction (FSI) simulations. A relative error of 6.7% and 19.8% was obtained for peak systolic (WSS

Focused ultrasound (FUS)-based viscoelastic imaging techniques using high frame rate (HFR) ultrasound to track tissue displacement can be used for mechanistic monitoring of FUS neuromodulation. However, a majority of techniques avoid imaging during the active push transmit (interleaved or postpush acquisitions) to mitigate ultrasound interference, which leads to missing temporal information of ultrasound effects when FUS is being applied. Furthermore, critical for clinical translation, use of both axial steering and real-time (1 s) capabilities for optimizing acoustic parameters for tissue engagement are largely missing. In this study, we describe a method of noninterleaved, single Vantage imaging displacement within an active FUS push with simultaneous axial steering and real-time capabilities using a single ultrasound acquisition machine. Results show that the pulse sequence can track micron-sized displacements using frame rates determined by the calculated time-of-flight (TOF), without interleaving the FUS pulses and imaging acquisition. Decimation by 3-7 frames increases signal-to-noise ratio (SNR) by 15.09±7.03 dB. Benchmarking tests of CUDA-optimized code show increase in processing speed of 35- and 300-fold in comparison with MATLAB parallel processing GPU and CPU functions, respectively, and we can estimate displacement from steered push beams ±10 mm from the geometric focus. Preliminary validation of displacement imaging in humans shows that the same driving pressures led to variable nerve engagement, demonstrating important feedback to improve transducer coupling, FUS incident angle, and targeting. Regarding the use of our technique for neuromodulation, we found that FUS altered thermal perception of thermal pain by 0.9643 units of pain ratings in a single trial. Additionally, 5 [Formula: see text] of nerve displacement was shown in on-target versus off-target sonications. The initial feasibility in healthy volunteers warrants further study for potential clinical translation of FUS for pain suppression.

The emergence of new ultrasound technologies has improved our understanding of the brain functions and offered new opportunities for the treatment of brain diseases. Ultrasound has become a valuable tool in preclinical animal and clinical studies as it not only provides information about the structure and function of brain tissues but can also be used as a therapy alternative for brain diseases. High-resolution cerebral flow images with high sensitivity can be acquired using novel functional ultrasound and super-resolution ultrasound imaging techniques. The noninvasive treatment of essential tremors has been clinically approved and it has been demonstrated that the ultrasound technology can revolutionize the currently existing treatment methods. Microbubble-mediated ultrasound can remotely open the blood-brain barrier enabling targeted drug delivery in the brain. More recently, ultrasound neuromodulation received a great amount of attention due to its noninvasive and deep penetration features and potential therapeutic benefits. This review provides a thorough introduction to the current state-of-the-art research on brain ultrasound and also introduces basic knowledge of brain ultrasound including the acoustic properties of the brain/skull and engineering techniques for ultrasound. Ultrasound is expected to play an increasingly important role in the diagnosis and therapy of brain diseases.

Neurons of the peripheral nervous system (PNS) are tasked with diverse roles, from encoding touch, pain, and itch to interoceptive control of inflammation and organ physiology. Thus, technologies that allow precise control of peripheral nerve activity have the potential to regulate a wide range of biological processes. Noninvasive modulation of neuronal activity is an important translational application of focused ultrasound (FUS). Recent studies have identified effective strategies to modulate brain circuits; however, reliable parameters to control the activity of the PNS are lacking. To develop robust noninvasive technologies for peripheral nerve modulation, we employed targeted FUS stimulation and electrophysiology in mouse ex vivo skin-saphenous nerve preparations to record the activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS pulses reliably and repeatedly evoked one-to-one action potentials in all peripheral neurons recorded. Interestingly, when neurons were classified based on neurophysiological properties, we identified a discrete range of FUS parameters capable of exciting all neuronal classes, including myelinated A fibers and unmyelinated C fibers. Peripheral neurons were excited by FUS stimulation targeted to either cutaneous receptive fields or peripheral nerves, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation. FUS elicited action potentials with millisecond latencies compared with electrical stimulation, suggesting ion channel–mediated mechanisms. Indeed, FUS thresholds were elevated in neurons lacking the mechanically gated channel PIEZO2. Together, these results demonstrate that transcutaneous FUS drives peripheral nerve activity by engaging intrinsic mechanotransduction mechanisms in neurons [B. U. Hoffman, PhD thesis, (2019)].

Optogenetics has revolutionized the capability of controlling genetically modified neurons in vitro and in vivo and has become an indispensable neuroscience tool. Using light as a probe for selective neuronal activation or inhibition and as a means to read out neural activity has dramatically enhanced our understanding of complex neural circuits. However, a common limitation of optogenetic studies to date is their invasiveness and spatiotemporal range. Direct viral injections into the brain tissue along with implantation of optical fibers and recording electrodes can disrupt the neuronal circuitry and cause significant damage. Conventional approaches are spatially limited around the site of the direct injection and insufficient in examining large networks throughout the brain. Lastly, optogenetics is currently not easily scalable to large animals or humans. Here, we demonstrate that optogenetic excitation can be achieved entirely non-invasively through the intact skull in mice. Using a needle-free combination of focused ultrasound-mediated viral delivery and extracorporeal illumination with red light, we achieved selective neuronal activation at depths up to 4 mm in the murine brain, confirmed through cFos expression and electrophysiology measurements within the treated areas. Ultrasound treatment significantly reduced freezing time during recall in fear conditioning experiments, but remote light exposure had a moderate effect on the freezing behavior of mice treated with viral vectors. The proposed method has the potential to open new avenues of studying, but also stimulating, neuronal networks, in an effort to elucidate normal or dysfunctional brain activity and treat neurological diseases. Finally, the same non-invasive methodology could be combined with gene therapy and applied to other organs, such as the eye and the heart.

Myocardial Elastography (ME) is an ultrasound-based strain imaging method. A survival canine model (n=11) was employed to investigate ME’s ability to image myocardial infarction (MI) formation and recovery. Infarcts were generated by ligation of the left anterior descending coronary artery. Canines were survived and imaged for four days (n=7) or four weeks (n=4), allowing sufficient time for recovery via collateral perfusion. A radial strain-based metric, percentage of healthy myocardium by strain (PHM(ε)), was developed as a marker for healthy myocardial tissue. PHM(ε) was strongly linearly correlated with actual infarct size as determined by gross pathology (R(2) = 0.80). Mean PHM(ε) was reduced 1–3 days postinfarction (p

Purpose: Pancreatic ductal adenocarcinoma (PDA) is a common, deadly cancer that is challenging both to diagnose and to manage. Its hallmark is an expansive, desmoplastic stroma characterized by high mechanical stiffness. In this study, we sought to leverage this feature of PDA for two purposes: differential diagnosis and monitoring of response to treatment. Experimental Design: Harmonic motion imaging (HMI) is a functional ultrasound technique that yields a quantitative relative measurement of stiffness suitable for comparisons between individuals and over time. We used HMI to quantify pancreatic stiffness in mouse models of pancreatitis and PDA as well as in a series of freshly resected human pancreatic cancer specimens. Results: In mice, we learned that stiffness increased during progression from preneoplasia to adenocarcinoma and also effectively distinguished PDA from several forms of pancreatitis. In human specimens, the distinction of tumors versus adjacent pancreatitis or normal pancreas tissue was even more stark. Moreover, in both mice and humans, stiffness increased in proportion to tumor size, indicating that tuning of mechanical stiffness is an ongoing process during tumor progression. Finally, using a brca2–mutant mouse model of PDA that is sensitive to cisplatin, we found that tissue stiffness decreases when tumors respond successfully to chemotherapy. Consistent with this observation, we found that tumor tissues from patients who had undergone neoadjuvant therapy were less stiff than those of untreated patients. Conclusions: These findings support further development of HMI for clinical applications in disease staging and treatment response assessment in PDA.

Temperature measurements with thin thermocouples embedded in ultrasound fields are strongly subjected to a viscous heating artifact (VHA). The artifact contribution decays over time; therefore, it can be minimized at late temperature readings. However, previous studies have failed to demonstrate a rigorous method for determining the optimal time point at which the artifact contribution is negligible. In this study, we present an iterative processing method based on successive curve fittings using an artifact-independent model. The fitting starting point moves at each iteration until the maximum $R^{2}$ indicates where the viscous heating is minimum. A solution of the bioheat transfer equation is used to account for blood perfusion, thus enabling in vivo measurements. Three T-type thermocouples with different diameters and sensitivities were assessed in an excised canine liver and in the mouse brain in vivo . We found that the artifact constitutes up to 81% ± 5% of wire thermocouple readings. The best-fit time varied in the liver samples ( $n = 3$ ) from 0 to 3.335 ± 0.979 s and in the mouse brain ( $n = 5$ ) from 0 to 0.498 ± 0.457 s at variable experimental conditions, which clearly demonstrates the need of the method for finding the appropriate starting time point of the fit. This study introduces a statistical method to determine the best time to fit a curve that can back-estimate temperature in tissues under ultrasound exposure using thermocouples. This method allows temperature evaluation in vivo and in vitro during a validation and safety assessment of a wide range of therapeutic and diagnostic ultrasound modalities.

Introduction: Electromechanical wave imaging (EWI) is a new ultrasound-based technique that can noninvasively map myocardial activation of all four chambers at high temporal resolution (500-2000 frames per second). Localization of atrial and ventricular arrhythmias with 3D-rendered EWI maps has been demonstrated and validated in heart rhythm disorders using multiple 2D echocardiographic views at different heartbeats. Objective: The objective of this study was to assess the in vivo performance of single heartbeat 3D EWI using coherent compounding of high volume-rate 3D anatomical echocardiography. Methods: A 32x32-element matrix array (Vermon, France) was connected to four synchronized research ultrasound scanners (Vantage 256, Verasonics, WA). High volume rate acquisitions were performed with diverging wave imaging at a compounded imaging rate of 840 volumes per second. Three healthy volunteers were imaged during normal sinus rhythm and one open chest canine was imaged during left ventricular pacing. Timing of zero-crossing of temporal inter-volume axial strain curves, corresponding to local onset of contraction, was determined for EWI activation. Results: A normal activation sequence was consistently observed in all volunteers (Figure 1). Early activation was mapped in the right atrium, followed by the left atrium, followed by the ventricles. Epicardial ventricular pacing was performed alternatively in the basal anterior-lateral region and in the apical anterior-septal region. The earliest activated region with 3D EWI was observed in the same location as the pacing electrode (Figure 1). Good agreement between 3D EWI and electro-anatomical mapping was obtained. Conclusions: Ultrasound-based full 3D EWI is capable of consistently mapping myocardial activation of the full heart in a single heartbeat in healthy volunteers noninvasively and is capable of identifying early activation during epicardial ventricular pacing in an open-chest canine.

Harmonic Motion Imaging (HMI) is an acoustic radiation-force (RF) based elasticity imaging technique which can be used to monitor focused ultrasound (FUS) induced ablation without interruption. Our group has previously demonstrated frequency domain filtering in removing FUS interference from imaging data. However, FUS interference becomes more pronounced at increased intensities and durations, and various imaging and tracking transducer configurations may cause an overlap in frequency spectra between the FUS and imaging pulse, making frequency domain filtering unsuitable. FUS-net, a CNN-based UNET autoencoder developed by our group, was previously shown to be able to reduce FUS artifacts in RF data acquired with single acoustic radiation force (ARF) pulses with a duration of 1 to 2 ms. In this study, FUS-net was applied to reduce FUS artifacts in an RF dataset acquired during HMI, with continuous wave oscillating pulses for a duration of 6s. The FUS-net model was trained and tested on beamformed RF data acquired from an in vivo breast cancer mouse model (N=3) and ex vivo human fibroadenoma breast tissue (N=1). Notch filtered beamformed RF data was used as ground truth. Displacement estimated from FUS-net processed data was compared to displacement estimated from notch filtered RF data. We showed less noise in the focal area of HMI displacement maps in the FUS-net filtered dataset as compared with frequency-based filtering, with a median coefficient of variation of 0.7 and 0.2 in displacement calculated from notch filtered RF data and FUS-net filtered data respectively.

Focused ultrasound (FUS) neuromodulation can induce therapeutic effects, such as decreasing perceived pain. To ensure safety during FUS and to better understand the role of cavitation during in vivo neuromodulation, factors influencing cavitation activity must be well understood. Tissue temperature may change between patients and heat may accumulate during therapy. Previous studies have shown a dependence of inertial cavitation thresholds on tissue shear modulus, which changes with temperature. Tissue temperature may therefore affect the intensity of cavitation in the tissue, yet this has not been well-characterized. This study sought to assess the relationship between tissue temperature and levels of cavitation under FUS in vivo. Cavitation was recorded during each sonication and analyzed using calculated passive cavitation images and cavitation doses. Two study designs were used to evaluate cavitation across tissue temperatures of 30-38°C. Our results showed that greater cavitation intensities occurred at higher tissue temperatures. Thus, temperature elevation and cavitation events should be monitored during FUS therapies.

Theranostic Ultrasound (TUS) is a novel modality for combined blood-brain barrier (BBB) opening and cavitation mapping within a single multielement imaging transducer. While previous work developing TUS has focused on understanding qualitative spatial agreement of a cavitation mapping strategy called power cavitation imaging with regions of BBB opening indicated on contrast-enhanced magnetic resonance images (MRI), the impact of short pulse lengths on signal intensity of power cavitation images has yet to be resolved. In this study we evaluated the impact of short pulse lengths on BBB opening volume, power cavitation imaging signal intensity, and BBB closure with a novel rapid beam steering protocol, enabling comparison of TUS parameters within the same animal. Significant increases in BBB opening volume, power cavitation image signal intensity and BBB closing duration were observed with pulse length. Given the faster BBB closing rate facilitated by short pulse transmits, along with the highly tunable nature of TUS sonication protocols, TUS could prove promising as an alternative to conventional focused ultrasound (FUS) configurations in the clinic.

An electromagnetic tracking system was used to track the position and orientation of an ultrasound imaging probe for the coregistration of multiple 2D left-ventricular strain images, which were computed via myocardial elastography. High frame-rate radiofrequency data were acquired from a healthy human volunteer in standard short-axis and apical views, and cross-correlation searches in the axial and lateral dimensions estimated the displacement and strain of the myocardial wall throughout ventricular systole. Positional coordinates and quarterions outputted by the electromagnetic sensor were then used to automatically translate and rotate each strain image for coregistration and simultaneous visualization of all acquisitions in a 3D grid. The final result is a patient-specific, anatomically accurate representation of systolic myocardial strain throughout the left ventricle.

Spatial resolution is typically prioritized over temporal in conventional echocardiography, which restricts the use of time-shifted based techniques at higher frame rates (FR). Electromechanical Wave Imaging (EWI) is an echocardiography-based modality that non-invasively maps the cardiac activation sequence at a high frame rate. At lower FRs, EWI strain curves have a different profile which constrains manual input and interpretation. In this study, we investigate how machine learning (ML) can assist in the accurate activation time estimation at low FR data. We show that with the use of a Random Forest classifier for automated EWI estimation, we successfully identify the normal, early activated basal septum in normal sinus rhythm lower FR cases ( $\mathrm{N}=4$ , FRs of 500 Hz, 250 Hz, 125 Hz) while maintaining the electromechanical activation propagation pattern over the rest of the myocardium. These findings indicate that ML can be used to generate accurate EWI activation maps with a more flexible FR range that includes clinical systems at low FRs, where manual processing would otherwise be impractical and/or inaccurate.

In addition to anatomical changes, mechanical properties and vascularization of breast tumors also alter in response to chemotherapeutic treatment. Early detection of non-responders to the treatment is critical for starting new chemotherapeutic regimens for improved tumor therapeutic response and overall survival of the patient. In this study, we used harmonic motion imaging which is an ultrasound elasticity imaging technique to monitor chemotherapy-induced stiffness changes of breast tumors in a xenograft mouse model. Alterations in tumor vasculature and angiogenesis were measured using functional ultrasound imaging that maps microvasculature with high spatial resolution and sensitivity. Preliminary results indicate the potential for combination of HMI and fUS for breast tumor chemotherapeutic response prediction and monitoring.

Traditional passive acoustic mapping (PAM) based on the time exposure acoustic (TEA) algorithm for focused ultrasound (FUS) brain therapy suffers from a poor axial resolution with a clinically-relevant frequency (< 3MHz) for transcranial imaging. Although some methods have been proposed to achieve better resolution, the real-time implementation is virtually impossible due to their computational complexity. Here, we developed a coherence factor (CF)-based PAM that can enhance the axial resolution with a low computational cost. In the proposed method, a CF across the delayed channel data was used as a weighting factor and the delay-and-sum with CF-weighting was repeated for every time sample and accumulated over the PAM integration time. The method was implemented on a Verasonics Vantage system with a GPU using CUDA. The CF exhibited better resolution than the TEA-PAM and comparable to the eigenspace-based robust Capon beamformer (EISRCB)-based PAM. The computational efficiency of CF-PAM was approximately 100 times higher than EISRCB-PAM, which in return enabled real-time monitoring. In the ex-vivo skull study, the CF-PAM successfully located the cavitation transcranially with improved resolution. While EISRCB required a careful parameter adjustment, the CF-PAM did not need any adjustment and provided reliable results throughout all the frames. In conclusion, this technique could help to monitor cavitation activities in real time during the sonication as part of several therapeutic ultrasound applications.

Focused Ultrasound (FUS) paired with systemically-introduced microbubbles (MB) is a widely-used noninvasive technique for transiently opening the Blood-Brain Barrier (BBB). FUS-BBBO is implicated in the treatment of several age-associated neuropathological disorders. The prevalence of these diseases in aged populations necessitates an investigation into and an understanding of the effect of age on the brain's response to FUS-BBBO. Here we show that advanced natural aging increases the brain's susceptibility to FUS-BBBO by increasing the volume of BBBO in response to equivalent sonication parameters. Additionally, the BBBO volume correlates positively with cavitation dose (CD). However, the extent of BBBO in response to cavitation is greater in two- compared to one-year old mice. Taken together, these results indicate an increased sensitivity in aged compared to younger mice, highlighting an important consideration in therapeutic planning.

Targeted drug delivery to enhance chemotherapy treatments in brain tumors can be achieved by opening the blood-brain barrier in a non-invasive, transient, localized, safe manner by using focused ultrasound (FUS) and microbubbles. However, as brain tumors are large and irregular structures, conventional single-element FUS methods are inefficient because they do not allow large covering. In this study, we numerically showed how 3D-printed acoustic holograms allow to correct the skull aberrations and to produce a large and localized focusing along a pediatric brain tumor. Several targeting configurations were numerically studied aiming to increase the covered tumor volume while reducing the off-target coverage. Fluid-like tissue simulations were performed using the k-space method. We used a 250-kHz single-element device in different configurations (ROC=110 mm, OD=110 or 132 mm, ID=0 or 44 mm), where the optimal setup with lens (OD=132 mm, ID=0 mm) provided a 16 % of tumor coverage compared to the 5 % obtained using the transducer without lens (OD=110 mm, ID=44 mm), while keeping a reduced 0.06 % of off-target coverage. The focal amplitude decreases 1.6 times accordingly to the focus spread. The estimated sonication duration and the number of microbubble injections can be reduced by 3.2 times, defining a time- and cost-efficient approach. The reported approach is simple, low-cost, and feasible to offering personalized, tumor-directed precision FUS treatment, to improve drug delivery for pediatric patients with brain tumors.

The emergence of new technologies has dramatically accelerated our understanding of the human brain function. Over the past decade, ultrasound has become a valuable tool in preclinical animal studies and clinical practice in many areas of the brain. This is because it can not only provide information about the structure and function of brain tissues but also constitute a potential treatment tool for brain disease. The recent FDA approval and success of ultrasound ablation techniques for essential tremors is an example of how ultrasound can revolutionize clinical practice. Similarly, recent brain ultrasound advances such as noninvasive blood–brain barrier (BBB) opening, ultrasound neuromodulation, and functional brain imaging have great potential to improve human patient outcomes in neurology and neurosurgery. Ultrasound is thus expected to play an increasingly important role in the diagnosis and treatment of brain disease in the upcoming years.

BackgroundOwing to its high spatial resolution and penetration depth, transcranial focused ultrasound stimulation (tFUS) is one of the most promising approaches to non-invasive neuromodulation. Identifying the impact of the stimulation waveform and endogenous neural activity on neuromodulation outcome is critical to harnessing the potential of tFUS.ObjectiveHere we tested a new form of tFUS where the amplitude of the ultrasonic waveform is modulated at a rate much slower than the operating frequency. Moreover, we sought to identify the relationship between pre-stimulation neural activity and the neuronal response to tFUS.MethodsWe applied three minutes of amplitude modulated (AM) tFUS at 40 Hz to the rat hippocampus while recording local field potentials (LFP) and multi-unit activity (MUA)from the sonicated region. To assess the role of AM, we also tested continuous-wave (CW) stimulation.ResultsAM tFUS reduced firing rate during and immediately after stimulation. On the other hand, CW tFUS produced an acute firing rate increase that was abolished after sonication. For both waveforms, firing rate changes were stronger in units exhibiting high baseline LFP power, particularly in the gamma band (30-250 Hz). The neuromodulatory effect was also influenced by the prevalence of sharp wave ripples (SWR) during the pre-stimulation period, with firing rates modulated by up to 33% at units showing frequent baseline SWR.ConclusionOur findings suggest that AM and CW tFUS produce qualitatively different neuronal outcomes, and that baseline rhythms may effectively “gate” the response to tFUS.