Branched Convolutional Neural Networks for Receiver Channel Recovery in High-Frame-Rate Sparse-Array Ultrasound Imaging.

For high-frame-rate ultrasound imaging, it remains challenging to implement on compact systems as a sparse imaging configuration with limited array channels. One key issue is that the resulting image quality is known to be mediocre not only because unfocused plane-wave excitations are used but also because grating lobes would emerge in sparse-array configurations. In this article, we present the design and use of a new channel recovery framework to infer full-array plane-wave channel datasets for periodically sparse arrays that operate with as few as one-quarter of the full-array aperture. This framework is based on a branched encoder-decoder convolutional neural network (CNN) architecture, which was trained using full-array plane-wave channel data collected from human carotid arteries (59 864 training acquisitions; 5-MHz imaging frequency; 20-MHz sampling rate; plane-wave steering angles between -15° and 15° in 1° increments). Three branched encoder-decoder CNNs were separately trained to recover missing channels after differing degrees of channelwise downsampling (2, 3, and 4 times). The framework's performance was tested on full-array and downsampled plane-wave channel data acquired from an in vitro point target, human carotid arteries, and human brachioradialis muscle. Results show that when inferred full-array plane-wave channel data were used for beamforming, spatial aliasing artifacts in the B-mode images were suppressed for all degrees of channel downsampling. In addition, the image contrast was enhanced compared with B-mode images obtained from beamforming with downsampled channel data. When the recovery framework was implemented on an RTX-2080 GPU, the three investigated degrees of downsampling all achieved the same inference time of 4 ms. Overall, the proposed framework shows promise in enhancing the quality of high-frame-rate ultrasound images generated using a sparse-array imaging setup.

Real-Time Speed-of-Sound Estimation in Vivo via Steered Plane Wave Ultrasound.

Speed-of-sound (SoS) is an intrinsic acoustic property of human tissues and has been regarded as a potential biomarker of tissue health. To foster the clinical use of this emerging biomarker in medical diagnostics, it is important for SoS estimates to be derived and displayed in real-time. Here, we demonstrate that concurrent global SoS estimation and B-mode imaging can be achieved live on a portable ultrasound scanner. Our innovation is hinged upon the design of a novel pulse-echo SoS estimation framework that is based on steered plane wave imaging. It has accounted for the effects of refraction and imaging depth when the medium SoS differs from the nominal value of 1540 m/s that is conventionally used in medical imaging. The accuracy of our SoS estimation framework was comparatively analyzed with through-transmit time-of-flight measurements in vitro on 15 custom agar phantoms with different SoS values (1508 to 1682 m/s) and in vivo on human calf muscles (N = 9; SoS range: 1560 to 1586 m/s). Our SoS estimation framework has a mean signed difference of, respectively, -0.6±2.3 m/s in vitro and -2.2±11.2 m/s in vivo relative to the reference measurements. Additionally, our real-time system prototype has yielded simultaneous SoS estimates and B-mode imaging at an average frame rate of 18.1 fps. Overall, by realizing real-time tissue SoS estimation with B-mode imaging, our innovation can foster the use of tissue SoS as a biomarker in medical ultrasound diagnostics.

Sensitivity improvement of subharmonic-based pressure measurement using phospholipid-coated monodisperse microbubbles.

The use of the subharmonic signal from microbubbles exposed to ultrasound is a promising safe and cost-effective approach for the non-invasive measurement of blood pressure. Achieving a high sensitivity of the subharmonic amplitude to the ambient overpressure is crucial for clinical applications. However, currently used microbubbles have a wide size distribution and diverse shell properties. This causes uncertainty in the response of the subharmonic amplitude to changes in ambient pressure, which limits the sensitivity. The aim of this study was to use monodisperse microbubbles to improve the sensitivity of subharmonic-based pressure measurements. With the same shell materials and gas core, we used a flow-focusing microfluidic chip and a mechanical agitation method to fabricate monodisperse (∼2.45-µm mean radius and 4.7 % polydisperse index) and polydisperse microbubbles (∼1.51-µm mean radius and 48.4 % polydisperse index), respectively. We varied the ultrasound parameters (i.e., the frequency, peak negative pressure (PNP) and pulse length), and found that there was an optimal excitation frequency (2.8 MHz) for achieving maximal subharmonic emission for monodisperse microbubbles, but not for polydisperse microbubbles. Three distinct regimes (occurrence, growth, and saturation) were identified in the response of the subharmonic amplitude to increasing PNP for both monodisperse and polydisperse microbubbles. For the polydisperse microbubbles, the subharmonic amplitude decreased either monotonically or non-monotonically with ambient overpressure, depending on the PNP. By contrast, for the monodisperse microbubbles, there was only a monotonic decrease at all PNPs. The maximum sensitivity (1.18 dB/kPa, R2 = 0.97) of the subharmonic amplitude to ambient overpressure for the monodisperse microbubbles was ∼6.5 times higher than that for the polydisperse microbubbles (0.18 dB/kPa, R2 = 0.88). These results show that monodisperse microbubbles can achieve a more consistent response of the subharmonic signal to changes in ambient overpressure and greatly improve the measurement sensitivity.

Transrectal ultrasound vector projectile imaging for time-resolved visualization of flow dynamics in the male urethra: A clinical pilot study.

BACKGROUND: Quantitative and comprehensive visualization of urinary flow dynamics in the urethra is crucial for investigating patient-specific mechanisms of lower urinary tract symptoms (LUTS). Although some methods can evaluate the global properties of the urethra, it is critical to assess the local information, such as the location of the responsible lesion and its interactions with urinary flow in relation to LUTS. This approach is vital for enhancing personalized and focal treatments. However, there is a lack of such diagnostic tools that can directly observe how the urethral shape and motion impact urinary flow in the urethra. PURPOSE: This study aimed to develop a novel transrectal ultrasound imaging modality based on the contrast-enhanced urodynamic vector projectile imaging (CE-UroVPI) framework and validate its clinical applicability for visualizing time-resolved flow dynamics in the urethra. METHODS: A new CE-UroVPI system was developed using a research-purpose ultrasound platform and a custom transrectal linear probe, and an imaging protocol for acquiring urodynamic echo data in male patients was designed. Thirty-four male patients with LUTS participated in this study. CE-UroVPI was performed to acquire ultrasound echo signals from the participant's urethra and urinary flow at various voiding phases (initiation, maintenance, and terminal). The ultrasound datasets were processed with custom software to visualize urinary flow dynamics and urethra tissue deformation. RESULTS: The transrectal CE-UroVPI system successfully visualized the time-resolved multidirectional urinary flow dynamics in the prostatic urethra during the initiation, maintenance, and terminal phases of voiding in 17 patients at a frame rate of 1250 fps. The maximum flow speed measured in this study was 2.5 m/s. In addition, when the urethra had an obstruction or an irregular partial deformation, the devised imaging modality visualized complex flow patterns, such as vortices and flow jets around the lesion. CONCLUSIONS: Our study findings demonstrate that the transrectal CE-UroVPI system developed in this study can effectively image fluid-structural interactions in the urethra. This new diagnostic technology has the potential to facilitate quantitative and precise assessments of urethral voiding functions and aid in the improvement of focal and effective treatments for patients with LUTS.

A GPU-Based, Real-Time Dealiasing Framework for High-Frame-Rate Vector Doppler Imaging.

Vector Doppler is well regarded as a potential way of deriving flow vectors to intuitively visualize complex flow profiles, especially when it is implemented at high frame rates. However, this technique's performance is known to suffer from aliasing artifacts. There is a dire need to devise real-time dealiasing solutions for vector Doppler. In this article, we present a new methodological framework for achieving aliasing-resistant flow vector estimation at real-time throughput from precalculated Doppler frequencies. Our framework comprises a series of compute kernels that have synergized: 1) an extended least squares vector Doppler (ELS-VD) algorithm; 2) single-instruction, multiple-thread (SIMT) processing principles; and 3) implementation on a graphical processing unit (GPU). Results show that this new framework, when executed on an RTX-2080 GPU, can effectively generate aliasing-free flow vector maps using high-frame-rate imaging datasets acquired from multiple transmit-receive angle pairs in a carotid phantom imaging scenario. Over the entire cardiac cycle, the frame processing time for aliasing-resistant vector estimation was measured to be less than 16 ms, which corresponds to a minimum processing throughput of 62.5 frames/s. In a human femoral bifurcation imaging trial with fast flow (150 cm/s), our framework was found to be effective in resolving two-cycle aliasing artifacts at a minimum throughput of 53 frames/s. The framework's processing throughput was generally in the real-time range for practical combinations of ELS-VD algorithmic parameters. Overall, this work represents the first demonstration of real-time, GPU-based aliasing-resistant vector flow imaging using vector Doppler estimation principles.

Ultrasound vector flow imaging during veno-arterial extracorporeal membrane oxygenation in a thoracic aorta model.

In veno-arterial extracorporeal membrane oxygenation (VA-ECMO) treatment, the mixing zone is a key hemodynamic factor that determines the efficacy of the treatment. This study aimed to evaluate the applicability of a novel ultrasound technique called vector flow imaging (VFI) for visualizing complex flow patterns in an aorta phantom under VA-ECMO settings. VFI experiments were performed to image aortic hemodynamics under VA-ECMO treatment simulated in an anthropomorphic thoracic aorta phantom using a pulsatile pump (cardiac output: 2.7 L/min) and an ECMO pump with two different flow rates, 0.35 L/min and 1.0 L/min. The cardiac cycle of hemodynamics in the ascending aorta, aortic arch, and descending aorta was visualized, and the spatio-temporal dynamics of flow vectors were analyzed. VFI successfully visualized dynamic flow patterns in the aorta phantom. When the flow rate of the ECMO pump increased, ECMO flow was more dominant than cardiac output in the diastole phase, and the speed of cardiac output was suppressed in the systole phase. Vortex flow patterns were also detected in the ascending aorta and the arch under both ECMO flow rate conditions. The VFI technique may provide new insights into aortic hemodynamics and facilitates effective and safe VA-ECMO treatment.

Deep-learning-assisted and GPU-accelerated vector Doppler imaging with aliasing-resistant velocity estimation.

Vector flow imaging is a diagnostic ultrasound modality that is suited for the visualization of complex blood flow dynamics. One popular way of realizing vector flow imaging at high frame rates over 1000 fps is to apply multi-angle vector Doppler estimation principles in conjunction with plane wave pulse-echo sensing. However, this approach is susceptible to flow vector estimation errors attributed to Doppler aliasing, which is prone to arise when a low pulse repetition frequency (PRF) is inevitably used due to the need for finer velocity resolution or because of hardware constraints. Existing dealiasing solutions tailored for vector Doppler may have high computational demand that makes them unfeasible for practical applications. In this paper, we present the use of deep learning and graphical processing unit (GPU) computing principles to devise a fast vector Doppler estimation framework that is resilient against aliasing artifacts. Our new framework works by using a convolutional neural network (CNN) to detect aliased regions in vector Doppler images and subsequently applying an aliasing correction algorithm only at these affected regions. The framework's CNN was trained using 15,000 in vivo vector Doppler frames acquired from the femoral and carotid arteries, including healthy and diseased conditions. Results show that our framework can perform aliasing segmentation with an average precision of 90 % and can render aliasing-free vector flow maps with real-time processing throughputs (25-100 fps). Overall, our new framework can improve the visualization quality of vector Doppler imaging in real-time.

In vivo evaluation of compliance mismatch on intimal hyperplasia formation in small diameter vascular grafts.

Small diameter synthetic vascular grafts have high failure rate due to the thrombosis and intimal hyperplasia formation. Compliance mismatch between the synthetic graft and native artery has been speculated to be one of the main causes of intimal hyperplasia. However, changing the compliance of synthetic materials without altering material chemistry remains a challenge. Here, we used poly(vinyl alcohol) (PVA) hydrogel as a graft material due to its biocompatibility and tunable mechanical properties to investigate the role of graft compliance in the development of intimal hyperplasia and in vivo patency. Two groups of PVA small diameter grafts with low compliance and high compliance were fabricated by dip casting method and implanted in a rabbit carotid artery end-to-side anastomosis model for 4 weeks. We demonstrated that the grafts with compliance that more closely matched with rabbit carotid artery had lower anastomotic intimal hyperplasia formation and higher graft patency compared to low compliance grafts. Overall, this study suggested that reducing the compliance mismatch between the native artery and vascular grafts is beneficial for reducing intimal hyperplasia formation.

Efficacy of ultrasound vector flow imaging in tracking omnidirectional pulsatile flow.

BACKGROUND: Ultrasound vector flow imaging (VFI) shows potential as an emerging non-invasive modality for time-resolved flow mapping. However, its efficacy in tracking multidirectional pulsatile flow with temporal resolvability has not yet been systematically evaluated because of the lack of an appropriate test protocol. PURPOSE: We present the first systematic performance investigation of VFI in tracking pulsatile flow in a meticulously designed scenario with time-varying, omnidirectional flow fields (with flow angles from 0° to 360°). METHODS: Ultrasound VFI was performed on a three-loop spiral flow phantom (4 mm diameter; 5 mm pitch) that was configured to operate under pulsatile flow conditions (10 ml/s peak flow rate; 1 Hz pulse rate; carotid pulse shape). The spiral lumen geometry was designed to simulate recirculatory flow dynamics observed in the heart and in curvy blood vessel segments such as the carotid bulb. The imaging sequence was based on steered plane wave pulsing (-10°, 0°, +10° steering angles; 5 MHz imaging frequency; 3.3 kHz interleaved pulse repetition frequency). VFI's pulsatile flow estimation performance and its ability to detect secondary flow were comparatively assessed against flow fields derived from computational fluid dynamics (CFD) simulations that included consideration of fluid-structure interactions (FSI). The mean percentage error (MPE) and the coefficient of determination (R2 ) were computed to assess the correspondence of the velocity estimates derived from VFI and CFD-FSI simulations. In addition, VFI's efficacy in tracking pulse waves was analyzed with respect to pressure transducer measurements made at the phantom's inlet and outlet. RESULTS: Pulsatile flow patterns rendered by VFI agreed with the flow profiles computed from CFD-FSI simulations (average MPE: -5.3%). The shape of the VFI-measured velocity magnitude profile generally matched the inlet flow profile. High correlation exists between VFI measurements and simulated flow vectors (lateral velocity: R2  = 0.8; axial velocity R2  = 0.89; beam-flow angle: R2  = 0.98; p < 0.0001 for all three quantities). VFI was found to be capable of consistently tracking secondary flow. It also yielded pulse wave velocity (PWV) estimates (5.72 ± 1.02 m/s) that, on average, are within 6.4% of those obtained from pressure transducer measurements (6.11 ± 1.15 m/s). CONCLUSION: VFI can consistently track omnidirectional pulsatile flow on a time-resolved basis. This systematic investigation serves well as a quality assurance test of VFI.

Time-Resolved Wall Shear Rate Mapping Using High-Frame-Rate Ultrasound Imaging.

In atherosclerosis, low wall shear stress (WSS) is known to favor plaque development, while high WSS increases plaque rupture risk. To improve plaque diagnostics, WSS monitoring is crucial. Here, we propose wall shear imaging (WASHI), a noninvasive contrast-free framework that leverages high-frame-rate ultrasound (HiFRUS) to map the wall shear rate (WSR) that relates to WSS by the blood viscosity coefficient. Our method measures WSR as the tangential flow velocity gradient along the arterial wall from the flow vector field derived using a multi-angle vector Doppler technique. To improve the WSR estimation performance, WASHI semiautomatically tracks the wall position throughout the cardiac cycle. WASHI was first evaluated with an in vitro linear WSR gradient model; the estimated WSR was consistent with theoretical values (an average error of 4.6% ± 12.4 %). The framework was then tested on healthy and diseased carotid bifurcation models. In both scenarios, key spatiotemporal dynamics of WSR were noted: 1) oscillating shear patterns were present in the carotid bulb and downstream to the internal carotid artery (ICA) where retrograde flow occurs; and 2) high WSR was observed particularly in the diseased model where the measured WSR peaked at 810 [Formula: see text] due to flow jetting. We also showed that WASHI could consistently track arterial wall motion to map its WSR. Overall, WASHI enables high temporal resolution mapping of WSR that could facilitate investigations on causal effects between WSS and atherosclerosis.

Faster calcium recovery and membrane resealing in repeated sonoporation for delivery improvement.

In sonoporation-based macromolecular delivery, repetitive microbubble cavitation in the bloodstream results in repeated sonoporation of cells or sonoporation of non-sonoporated neighboring cells (i.e., adjacent to the sonoporated host cells). The resealing and recovery capabilities of these two types of sonoporated cells affect the efficiency and biosafety of sonoporation-based delivery. Therefore, an improved understanding of the preservation of viability in these sonoporated cells is necessary. Using a customized platform for single-pulse ultrasound exposure (pulse length 13.33 µs, peak negative pressure 0.40 MPa, frequency 1.5 MHz) and real-time recording of membrane perforation and intracellular calcium fluctuations (using propidium iodide and Fluo-4 fluorescent probes, respectively), spatiotemporally controlled sonoporation was performed to administer first and second single-site sonoporations of a single cell or single-site sonoporation of a neighboring cell. Two distinct intracellular calcium changes, reversible and irreversible calcium fluctuations, were identified in cells undergoing repeat reversible sonoporation and in neighboring cells undergoing reversible sonoporation. In addition to an increased proportion of reversible calcium fluctuations that occurred with repeated sonoporation compared with that in the initial sonoporation, repeated sonoporation resulted in significantly shorter calcium fluctuation durations and faster membrane resealing than that produced by initial sonoporation. Similarly, compared with those in sonoporated host cells, the intracellular calcium fluctuation recovery and membrane perforation resealing times were significantly shorter in sonoporated neighboring cells. These results demonstrated that the function recovery and membrane resealing capabilities after a second sonoporation or sonoporation of neighboring cells were potentiated in the short term. This could aid in sustaining the long-term viability of sonoporated cells, therefore improving delivery efficiency and biosafety. This investigation provides new insight into the resealing and recovery capabilities in re-sonoporation of sonoporated cells and sonoporation of neighboring cells and can help develop safe and efficient strategies for sonoporation-based drug delivery.

Unfocused Field Analysis of a Density-Tapered Spiral Array for High-Volume-Rate 3-D Ultrasound Imaging.

Spiral array transducers with a sparse 2-D aperture have demonstrated their potential in realizing 3-D ultrasound imaging with reduced data rates. Nevertheless, their feasibility in high-volume-rate imaging based on unfocused transmissions has yet to be established. From a metrology standpoint, it is essential to characterize the acoustic field of unfocused transmissions from spiral arrays not only to assess their safety but also to identify the root cause of imaging irregularities due to the array's sparse aperture. Here, we present a field profile analysis of unfocused transmissions from a density-tapered spiral array transducer (256 hexagonal elements, 220- [Formula: see text] element diameter, and 1-cm aperture diameter) through both simulations and hydrophone measurements. We investigated plane- and diverging-wave transmissions (five-cycle, 7.5-MHz pulses) from 0° to 10° steering for their beam intensity characteristics and wavefront arrival time profiles. Unfocused firings were also tested for B-mode imaging performance (ten compounded angles, -5° to 5° span). The array was found to produce unfocused transmissions with a peak negative pressure of 93.9 kPa at 2 cm depth. All transmissions steered up to 5° were free of secondary lobes within 12 dB of the main beam peak intensity. All wavefront arrival time profiles were found to closely match the expected profiles with maximum root-mean-squared errors of [Formula: see text] for plane wave (PW) and [Formula: see text] for diverging wave. The B-mode images showed good spatial resolution with a penetration depth of 22 mm in PW imaging. Overall, these results demonstrate that the density-tapered spiral array can facilitate unfocused transmissions below regulatory limits (mechanical index: 0.034; spatial-peak, pulse-average intensity: 0.298 W/cm2) and with suppressed secondary lobes while maintaining smooth wavefronts.

Minimizing Image Quality Loss After Channel Count Reduction for Plane Wave Ultrasound via Deep Learning Inference.

High-frame-rate ultrasound imaging uses unfocused transmissions to insonify an entire imaging view for each transmit event, thereby enabling frame rates over 1000 frames per second (fps). At these high frame rates, it is naturally challenging to realize real-time transfer of channel-domain raw data from the transducer to the system back end. Our work seeks to halve the total data transfer rate by uniformly decimating the receive channel count by 50% and, in turn, doubling the array pitch. We show that despite the reduced channel count and the inevitable use of a sparse array aperture, the resulting beamformed image quality can be maintained by designing a custom convolutional encoder-decoder neural network to infer the radio frequency (RF) data of the nullified channels. This deep learning framework was trained with in vivo human carotid data (5-MHz plane wave imaging, 128 channels, 31 steering angles over a 30° span, and 62 799 frames in total). After training, the network was tested on an in vitro point target scenario that was dissimilar to the training data, in addition to in vivo carotid validation datasets. In the point target phantom image beamformed from inferred channel data, spatial aliasing artifacts attributed to array pitch doubling were found to be reduced by up to 10 dB. For carotid imaging, our proposed approach yielded a lumen-to-tissue contrast that was on average within 3 dB compared to the full-aperture image, whereas without channel data inferencing, the carotid lumen was obscured. When implemented on an RTX-2080 GPU, the inference time to apply the trained network was 4 ms, which favors real-time imaging. Overall, our technique shows that with the help of deep learning, channel data transfer rates can be effectively halved with limited impact on the resulting image quality.

Sonoporation of Immune Cells: Heterogeneous Impact on Lymphocytes, Monocytes and Granulocytes.

Microbubble-mediated ultrasound (MB-US) can be used to realize sonoporation and, in turn, facilitate the transfection of leukocytes in the immune system. Nevertheless, the bio-effects that can be induced by MB-US exposure on leukocytes have not been adequately studied, particularly for different leukocyte lineage subsets with distinct cytological characteristics. Here, we describe how that same set of MB-US exposure conditions would induce heterogeneous bio-effects on the three main leukocyte subsets: lymphocytes, monocytes and granulocytes. MB-US exposure was delivered by applying 1-MHz pulsed ultrasound (0.50-MPa peak negative pressure, 10% duty cycle, 30-s exposure period) in the presence of microbubbles (1:1 cell-to-bubble ratio); sonoporated and non-viable leukocytes were respectively labeled using calcein and propidium iodide. Flow cytometry was then performed to classify leukocytes into their corresponding subsets and to analyze each subset's post-exposure viability, sonoporation rate, uptake characteristics and morphology. Results revealed that, when subjected to MB-US exposure, granulocytes experienced the highest loss of viability (64.0 ± 11.0%) and the lowest sonoporation rate (6.3 ± 2.5%), despite maintaining their size and granularity. In contrast, lymphocytes exhibited the lowest loss of viability (20.9 ± 7.0%), while monocytes had the highest sonoporation rate (24.1 ± 13.6%). For these two sonoporated leukocyte subsets, their cell size and granularity were found to be reduced. Also, they exhibited graded levels of calcein uptake, whereas sonoporated granulocytes achieved only mild calcein uptake. These heterogeneous bio-effects should be accounted for when using MB-US and sonoporation in immunomodulation applications.

Quantitative Blood Flow Measurements in the Common Carotid Artery: A Comparative Study of High-Frame-Rate Ultrasound Vector Flow Imaging, Pulsed Wave Doppler, and Phase Contrast Magnetic Resonance Imaging.

V Flow is commercially developed by high-frame-rate ultrasound vector flow imaging. Compared to conventional color Doppler, V Flow is angle-independent and is capable of measuring both the magnitude and the direction of blood flow velocities. This paper aims to investigate the differences between V Flow and pulsed wave Doppler (PW) relative to phase contrast magnetic resonance imaging (PC-MRI), for the quantitative measurements of blood flow in common carotid arteries (CCA) and, consequently, to evaluate the accuracy of the new technique, V Flow. Sixty-four CCAs were measured using V Flow, PW, and PC-MRI. The maximum velocities, time-averaged mean (TAMEAN) velocities, and volume flow were measured using different imaging technologies. The mean error with standard deviation (Std), the median of absolute errors, and the r-values between V Flow and PC-MRI results for the maximum velocity, the TAMEAN velocity, and the volume flow measurements are {9.40% ± 14.91%; 11.84%; 0.84}, {21.52% ± 14.46%; 19.28%; 0.86}, and {-2.80% ± 14.01%; 10.38%; 0.7}, respectively, and are {53.44% ± 29.68%; 49.79%; 0.74}, {27.83% ± 31.60%; 23.83; 0.71}, and {21.01% ± 29.64%; 25.48%; 0.34}, respectively, for those between PW and PC-MRI. The boxplot, linear regression and Bland-Altman plots were performed for each comparison, which illustrated that the results measured via V Flow rather than via PW agreed more closely with those measured via PC-MRI.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.

An enhanced, rational model to study acoustic vaporization dynamics of a bubble encapsulated within a nonlinearly elastic shell.

Acoustic droplet vaporization (ADV) is a new approach to generate vapor bubbles that have potentially broad medical applications. ADV-generated bubbles can be used as contrast agents in acoustic imaging, as drug carriers to deliver drugs to particular targets, and also in embolotherapy, thermal therapy, and histotripsy. However, despite much progress, ADV dynamics have still not been well understood and properly modeled. In this paper, we present a theoretical study of ultrasound-induced evaporation of a droplet encapsulated by a shell. The main emphasis of this theoretical study is on a proper description of the supercritical state occurring after bubble collapse. For this purpose, an isentropic equation of state for a van der Waals gas is used to describe the bubble behavior in the supercritical state. Sensitivity of the vaporization process is investigated for different acoustic and geometrical parameters and mechanical properties of the shell. Results show that the value of the minimum pressure required for direct vaporization (without any oscillatory behavior) depends on shell elasticity and initial size of the droplet, especially at high frequencies (greater than 2[MHz]). Moreover, it has been shown that applying an acoustic wave with proper phase such that thermal equilibrium of the bubble temperature with the surrounding liquid is attained, results in direct vaporization at lower acoustic pressure.

Ultrasound-Mediated Drug Delivery: Sonoporation Mechanisms, Biophysics, and Critical Factors.

Sonoporation, or the use of ultrasound in the presence of cavitation nuclei to induce plasma membrane perforation, is well considered as an emerging physical approach to facilitate the delivery of drugs and genes to living cells. Nevertheless, this emerging drug delivery paradigm has not yet reached widespread clinical use, because the efficiency of sonoporation is often deemed to be mediocre due to the lack of detailed understanding of the pertinent scientific mechanisms. Here, we summarize the current observational evidence available on the notion of sonoporation, and we discuss the prevailing understanding of the physical and biological processes related to sonoporation. To facilitate systematic understanding, we also present how the extent of sonoporation is dependent on a multitude of factors related to acoustic excitation parameters (ultrasound frequency, pressure, cavitation dose, exposure time), microbubble parameters (size, concentration, bubble-to-cell distance, shell composition), and cellular properties (cell type, cell cycle, biochemical contents). By adopting a science-backed approach to the realization of sonoporation, ultrasound-mediated drug delivery can be more controllably achieved to viably enhance drug uptake into living cells with high sonoporation efficiency. This drug delivery approach, when coupled with concurrent advances in ultrasound imaging, has potential to become an effective therapeutic paradigm.

Improving temporal stability of stable cavitation activity of circulating microbubbles using a closed-loop controller based on pulse-length regulation.

Stable cavitation (SC) has shown great potential for novel therapeutic applications. The spatiotemporal distribution of the SC activity of microbubbles circulating in a target region is not only correlated with the uniformity of treatment, but also with some undesirable effects. Therefore, it is important to achieve controllable and desirable SC activity in target regions for improved therapeutic efficiency and biosafety. This study proposes a closed-loop feedback controller based on pulse length (PL) regulation to improve the temporal stability of SC activity. Microbubbles circulating in a physiological flowing phantom were exposed to a 1 MHz focused transducer. The SC signals produced were initially received by another 7.5 MHz plane transducer, followed by high-speed signal acquisition and real-time processing. Based on the real-time-measured SC intensity excited by the current acoustic pulse, the proposed closed-loop feedback controller used three proportional coefficients to regulate the peak negative pressure (PNP) and PL of the next acoustic pulse during the acceleration and stable stages, respectively. The results show that the rise time and the temporal stability of the SC intensity of the microbubbles circulating in these two stages were improved significantly by the optimized proportional coefficients used in the proposed controller. Importantly, when compared with the traditional closed-loop feedback controller based on PNP regulation, the proposed closed-loop feedback controller based on PL regulation reduced the probability of a transition between stable and inertial cavitation, thus avoiding the risk of disadvantageous bioeffects in practical applications. These results demonstrate the effectiveness of the proposed PL-based closed-loop feedback controller and provide a feasible strategy for realization of controllable cavitation activity in applications.

Vascular Imaging in Small Animals Using Clinical Ultrasound Scanners.

Conventional ultrasound with frequency (2-15 MHz) has been a global diagnostic and therapeutic tool in clinical medicine, and high-frequency ultrasound (>30 MHz) has been a powerful investigative device for preclinical studies such as cardiovascular research. In this chapter, we describe the use of conventional ultrasound with a 2.5-10 MHz transducer as an investigative device for the measurement/detection of blood flow in rabbit model. The chapter will describe the procedures for the preparation of sonographer, imaging locations, and the details of the rabbits used as well as detailed imaging steps for the preoperative, immediately after operation, and postoperative follow-up ultrasound for vascular surgery, using a vascular graft implantation as an example. We also provide useful notes to avoid pitfalls for successful imaging. The overall goal of this chapter is to deliver the steps in using low-cost, non-invasive, and highly versatile clinical ultrasound imaging in preclinical small animal testing.