Focused ultrasound stimulation of infralimbic cortex attenuates reinstatement of methamphetamine-induced conditioned place preference in rats.

Methamphetamine (MA) use disorder poses significant challenges to both the affected individuals and society. Current non-drug therapies like transcranial direct-current stimulation and transcranial magnetic stimulation have limitations due to their invasive nature and limited reach to deeper brain areas. Transcranial focused ultrasound (FUS) is gaining attention as a noninvasive option with precise spatial targeting, able to affect deeper areas of the brain. This research focused on assessing the effectiveness of FUS in influencing the infralimbic cortex (IL) to prevent the recurrence of MA-seeking behavior, using the conditioned place preference (CPP) method in rats. The study involved twenty male Sprague-Dawley rats. Neuronal activation by FUS was first examined via electromyography (EMG). Rats received alternately with MA or saline, and confined to one of two distinctive compartments in a three compartment apparatus over a 4-day period. After CPP test, extinction, the first reinstatement, and extinction again, FUS was applied to IL prior to the second MA priming-induced reinstatement. Safety assessments were conducted through locomotor and histological function examinations. EMG data confirmed the effectiveness of FUS in activating neurons. Significant attenuation of reinstatement of MA CPP was found, along with successful targeting of the IL region, confirmed through acoustic field scanning, c-Fos immunohistochemistry, and Evans blue dye staining. No damage to brain tissue or impaired locomotor activity was observed. The results of the study indicate that applying FUS to the IL markedly reduced the recurrence of MA seeking behavior, without harming brain tissue or impairing motor skills. This suggests that FUS could be a promising method for treating MA use disorder, with the infralimbic cortex being an effective target for FUS in preventing MA relapse.

Feasibility of in vitro calcification plaque disruption using ultrasound-induced microbubble inertial cavitation.

Percutaneous transluminal coronary angioplasty (PTCA) is a clinical method in which plaque-narrowed arteries are widened by inflating an intravascular balloon catheter. However, PTCA remains challenging to apply in calcified plaques since the high pressure required for achieving a therapeutic outcome can result in balloon rupture, vessel rupture, and intimal dissection. To address the problem with PTCA, we hypothesized that a calcified plaque can be disrupted by microbubbles (MBs) inertial cavitation induced by ultrasound (US). This study proposed a columnar US transducer with a novel design to generate inertial cavitation at the lesion site. Experiments were carried out using tubular calcification phantom to mimic calcified plaques. After different parameters of US + MBs treatment (four types of MBs concentration, five types of cycle number, and three types of insonication duration; n = 4 in each group), inflation experiments were performed to examine the efficacy of cavitation for a clinically used balloon catheter. Finally, micro-CT was used to investigate changes in the internal structure of the tubular plaster phantoms. The inflation threshold of the untreated tubular plaster phantoms was > 11 atm, and this was significantly reduced to 7.4 ± 0.7 atm (p = 5.2E-08) using US-induced MBs inertial cavitation at a treatment duration of 20 min with an acoustic pressure of 214 kPa, an MBs concentration of 4.0 × 108 MBs/mL, a cycle number of 100 cycles, and a pulse repetition frequency of 100 Hz. Moreover, micro-CT revealed internal damage in the tubular calcification phantom, demonstrating that US-induced MBs inertial cavitation can effectively disrupt calcified plaques and reduce the inflation threshold of PTCA. The ex vivo histopathology results showed that the endothelium of pig blood vessels remained intact after the treatment. In summary, the results show that US-induced MBs inertial cavitation can markedly reduce the inflation threshold in PTCA without damaging blood vessel endothelia, indicating the potential of the proposed treatment method.

The relationship between surface drug distribution of Dox-loaded microbubbles and drug release/cavitation behaviors with ultrasound.

Ultrasound (US)-triggered microbubbles (MBs) drug delivery is a promising tool for noninvasive and localized therapy. Several studies have shown the potential of drug-loaded MBs to boost the delivery of therapeutic substances to target tissue effectively. Nevertheless, little is known about the surface payload distribution affecting the cavitation activity and drug release behavior of the drug-loaded MBs. In this study, we designed a common chemodrug (Doxorubicin, Dox)-loaded MB (Dox-MBs) and regulated the payload distribution as uniform or cluster onto the outer surface of MBs. The Dox distribution on the MB shells was assessed by confocal fluorescence microscopic imaging. The acoustic properties of the Dox-MBs with different Dox distributions were evaluated by their acoustic stability and cavitation activities. The payload release and the fragments from Dox-MBs in response to different US parameters were measured and visualized by column chromatography and cryo-electron microscopy, respectively. By amalgamating these methodologies, we found that stable cavitation was sufficient for triggering uniform-loaded MBs to release their payload, but stable cavitation and inertial cavitation were required for cluster-loaded MBs. The released substances included free Dox and Dox-containing micelle/liposome; their portions depended on the payload distribution, acoustic pressure, cycle number, and sonication duration. Furthermore, we also revealed that the Dox-containing micelle/liposome in cluster-loaded MBs had the potential for multiple drug releases upon US sonication. This study compared uniform-loaded MBs and cluster-loaded MBs to enhance our comprehension of drug-loaded MBs mediated drug delivery.

Effects of canagliflozin on cardiac remodeling and hemodynamic parameters in patients with type 2 diabetes mellitus.

Sodium-glucose cotransporter type 2 (SGLT2) inhibitors have demonstrated to reduce cardiovascular risk in patients with type 2 diabetes mellitus (T2DM) in large trials independent of glycemic control. The mechanisms of this cardioprotective property remain uncertain. Evidence suggests positive hemodynamic changes and favorable cardiac remodeling contributing to the clinical outcomes but results were conflicting. We aim to investigate the potential impact on hemodynamic parameters, cardiac structure and functions. This prospective observational study included T2DM patients receiving canagliflozin 100 mg per day in addition to their antidiabetic treatment. We analyzed hemodynamic parameters assessed by echocardiographic measurements and impedance cardiography (ICG) to evaluate systolic and diastolic functions from baseline to 24 weeks after treatment. A total of 47 patients (25 males and 22 females) averaging 64.6 ± 10.9 years had a significant reduction in HbA1c, body weight, and systolic blood pressure. Hematocrit increased significantly, while NT-proBNP remained unchanged. E/e', left atrium (LA) volume, and LA stiffness were reduced, while left ventricle (LV) global longitudinal strain (GLS) and LA strain rates increased at 24 weeks by conventional and speckle tracking echocardiography. LV mass and ejection fraction showed no differences. ICG suggested significant improvement in hemodynamic parameters with increased stroke volume index and cardiac output index and decreased systemic vascular resistance index at 12 and 24 weeks. Canagliflozin improved hemodynamic parameters and had a favorable impact on LA and LV reverse remodeling. These changes may explain the beneficial effect on cardiovascular outcomes in large clinical trials.

Electrowetting-driven liquid lens for ultrasound: Enabling controllable focal length and flexible beam steering.

Focused ultrasound is an increasingly popular non-invasive treatment modality. Still, its fixed focal point requires an array ultrasound transducer or scanning system to cover different therapeutic scenarios. To address this limitation, we developed an electrically-controlled liquid lens that enables dynamic beam focusing and steering of the incident plane ultrasound beam. The lens was carefully optimized for low-energy attenuation and low-voltage driving. We evaluated the performance of the lens using a homemade 5.5-MHz planar transducer with a 7.5-mm aperture. Our results demonstrate that the planar ultrasound beam can be adjusted to a focused beam with a focal length from 27 mm to 32 mm within 1 s by increasing the electric input (0-60 V) to the lens. Additionally, the beam angle of the ultrasound is tunable from -5 to 5° by adjusting the charge distribution on the lens. Our design enables real-time, fast-response, on-demand changing of focal length and beam angle for a single-element planar transducer. Our study presents a promising technology for altering the ultrasound beam of a planar single-element transducer for different ultrasound applications. The development of this electrically-controlled liquid lens has the potential to enhance the efficacy of focused ultrasound treatment and improve patient outcomes.

Oxygen-loaded microbubble-mediated sonoperfusion and oxygenation for neuroprotection after ischemic stroke reperfusion.

BACKGROUND: Ischemic stroke-reperfusion (S/R) injury is a crucial issue in the protection of brain function after thrombolysis. The vasodilation induced by ultrasound (US)-stimulated microbubble cavitation has been applied to reduce S/R injury through sonoperfusion. The present study uses oxygen-loaded microbubbles (OMBs) with US stimulation to provide sonoperfusion and local oxygen therapy for the reduction of brain infarct size and neuroprotection after S/R. METHODS: The murine S/R model was established by photodynamic thrombosis and thrombolysis at the remote branch of the anterior cerebral artery. In vivo blood flow, partial oxygen pressure (pO2), and brain infarct staining were examined to analyze the validity of the animal model and OMB treatment results. The animal behaviors and measurement of the brain infarct area were used to evaluate long-term recovery of brain function. RESULTS: The percentage of blood flow was 45 ± 3%, 70 ± 3%, and 86 ± 2% after 60 min stroke, 20 min reperfusion, and 10 min OMB treatment, respectively, demonstrating sonoperfusion, and the corresponding pO2 level was 60 ± 1%, 76 ± 2%, and 79 ± 4%, showing reoxygenation. After 14 days of treatment, a 87 ± 3% reduction in brain infarction and recovery of limb coordination were observed in S/R mice. The expression of NF-κB, HIF-1α, IL-1ß, and MMP-9 was inhibited and that of eNOS, BDNF, Bcl2, and IL-10 was enhanced, indicating activation of anti-inflammatory and anti-apoptosis responses and neuroprotection. Our study demonstrated that OMB treatment combines the beneficial effects of sonoperfusion and local oxygen therapy to reduce brain infarction and activate neuroprotection to prevent S/R injury.

Selective Activation of Cells by Piezoelectric Molybdenum Disulfide Nanosheets with Focused Ultrasound.

An accurate method for neural stimulation within the brain could be very useful for treating brain circuit dysfunctions and neurological disorders. With the aim of developing such a method, this study investigated the use of piezoelectric molybdenum disulfide nanosheets (MoS2 NS) to remotely convert ultrasound energy into localized electrical stimulation in vitro and in vivo. The application of ultrasound to cells surrounding MoS2 NS required only a single pulse of 2 MHz ultrasound (400 kPa, 1,000,000 cycles, and 500 ms pulse duration) to elicit significant responses in 37.9 ± 7.4% of cells in terms of fluxes of calcium ions without detectable cellular damage. The proportion of responsive cells was mainly influenced by the acoustic pressure, number of ultrasound cycles, and concentration of MoS2 NS. Tests using appropriate blockers revealed that voltage-gated membrane channels were activated. In vivo data suggested that, with ultrasound stimulation, neurons closest to the MoS2 NS were 3-fold more likely to present c-Fos expression than cells far from the NS. The successful activation of neurons surrounding MoS2 NS suggests that this represents a method with high spatial precision for selectively modulating one or several targeted brain circuits.

Application of Ultrasound to Enhancing Stem Cells Associated Therapies.

Pluripotent stem cell therapy exhibits self-renewal capacity and multi-directional differentiation potential and is considered an important regenerative approach for the treatment of several diseases. However, insufficient cell transplantation efficiency, uncontrollable differentiation, low cell viability, and difficult tracing limit its clinical applications and treatment outcome. Ultrasound (US) has mechanical, cavitation, and thermal effects that can produce different biological effects on organs, tissues, and cells. US can be combined with different US-responsive particles for enhanced physical-chemical stimulation and drug delivery. In the meantime, US also can provide a noninvasive and harmless imaging modality for deep tissue in vivo. An in-depth evaluation of the role and mechanism of action of US in stem cell therapy would enhance understanding of US and encourage research in this field. In this article, we comprehensively review progress in the application of US alone and combined with US-responsive particles for the promotion of proliferation, differentiation, migration, and in vivo detection of stem cells and the potential clinical applications.

Preventing ischemia-reperfusion injury by acousto-mechanical local oxygen delivery.

Ischemia-reperfusion (I/R) injury is a pathological process that causes vascular damage and dysfunction which increases recurrence and/or mortality in myocardial infarction, ischemic stroke, and organ transplantation. We hypothesized that ultrasound-stimulated oxygen-loaded microbubble (O2-MB) cavitation would enhance mechanical force on endothelium and simultaneously release oxygen locally at the targeted vessels. This cooperation between biomechanical and biochemical stimuli might modulate endothelial metabolism, providing a potential clinical approach to the prevention of I/R injury. Murine hindlimb and cardiac I/R models were used to demonstrate the feasibility of injury prevention by O2-MB cavitation. Increased mechanical force on endothelium induced eNOS-activated vasodilation and angiogenesis to prevent re-occlusion at the I/R vessels. Local oxygen therapy increased endothelial oxygenation that inhibited HIF-1α expression, increased ATP generation, and activated cyclin D1 for cell repair. Moreover, a decrease in interstitial H2O2 level reduced the expression of caspase3, NFκB, TNFα, and IL6, thus ameliorating inflammatory responses. O2-MB cavitation showed efficacy in maintaining cardiac function and preventing myocardial fibrosis after I/R. Finally, we present a potential pathway for the modulation of endothelial metabolism by O2-MB cavitation in relation to I/R injury, wound healing, and vascular bioeffects.

Enhanced sonodynamic therapy by carbon dots-shelled microbubbles with focused ultrasound.

Sonodynamic therapy involving the non-invasive and local generation of lethal reactive oxygen species (ROS) via ultrasound (US) with sonosensitizers has been proposed as an emerging tumor therapy strategy. However, such therapy is usually associated with inertial cavitation and unnecessary damage to healthy tissue because current sonosensitizers have insufficient sensitivity to US. Here, we report the use of a new proposed sonosensitizer, carbon dots (C-dots), to assemble microbubbles with a gas core (C-dots MBs). As the C-dots were directly integrated into the MB shell, they could effectively absorb the energy of inertial cavitation and transfer it to ROS. Our results revealed the appearance of 1O2, •OH, and H2O2 after US irradiation of C-dots MBs. In in vitro experiments, treatment with C-dots MBs plus US induced lipid peroxidation, elevation of intracellular ROS, and apoptosis in 32.5%, 45.3%, and 50.1% of cells respectively. In an animal solid tumor model, treatment with C-dots MBs plus US resulted in a 3-fold and 2.5-fold increase in the proportion of ROS-damaged cells and apoptotic cells, respectively, compared to C-dots MBs alone. These results will pave the way for the design of novel multifunctional sonosensitizers for SDT tumor therapy.

Genetically encoded mediators for sonogenetics and their applications in neuromodulation.

Sonogenetics is an emerging approach that harnesses ultrasound for the manipulation of genetically modified cells. The great penetrability of ultrasound waves enables the non-invasive application of external stimuli to deep tissues, particularly advantageous for brain stimulation. Genetically encoded ultrasound mediators, a set of proteins that respond to ultrasound-induced bio-effects, play a critical role in determining the effectiveness and applications of sonogenetics. In this context, we will provide an overview of these ultrasound-responsive mediators, delve into the molecular mechanisms governing their response to ultrasound stimulation, and summarize their applications in neuromodulation.

Enhancing Doxorubicin Delivery in Solid Tumor by Superhydrophobic Amorphous Calcium Carbonate-Doxorubicin Silica Nanoparticles with Focused Ultrasound.

The current approach of delivering chemotherapy via pH-sensitive amorphous calcium carbonate-doxorubicin silica nanoparticles (ADS NPs) faces the challenge of insufficient drug dose due to drug instability within the bloodstream and poor tumor penetration. To overcome these long-standing obstacles, we proposed a superhydrophobic coating on the surface of the ADS NPs that could be easily modified via fluorination (ADSF NPs). The surface of fluorinated ADS NPs was further modified with a phospholipid layer to reduce aggregation and improve biocompatibility (ADSFL NPs). The contact angle and mean size of ADSFL NPs were 30.2 ± 4.4° and 353.1 ± 54.2 nm, respectively. The superhydrophobic layer generated interfacial nanobubbles on the outer shell of the NPs that reduced water-induced leakage of doxorubicin (DOX) sevenfold compared with the uncoated group and induced a cavitation effect upon ultrasound (US) sonication. Moreover, release of DOX from the ADSFL NPs could be triggered by US, and this release was further improved 1.6-fold in acidic aqueous conditions, indicating that the ADSFL NPs retained pH responsiveness. Enhanced sonography contrast and histological examination demonstrated that US could trigger cavitation activities from ADSFL NPs in vivo to induce vessel disruption and enhance the fluorescence intensity of DOX within the tumor region threefold under US imaging guidance compared with the ADSFL NPs-only group.

State-of-the-art of ultrasound-triggered drug delivery from ultrasound-responsive drug carriers.

INTRODUCTION: Delivering sufficient therapeutics at the target site without off-target effects is a major goal of drug delivery technology innovation. Among the established methods, ultrasound (US) with US-responsible carriers holds great promise and demonstrates on-demand delivery of a variety of functional substances with spatial precision of several millimeters in deep-seated tissues in animal models and humans. These properties have motivated several explorations of US with US responsible-responsible carriers as a modality for neuromodulation and the treatment of various diseases, such as stroke and cancer. AREAS COVERED: We briefly discuss three specific mechanisms that enhance in vivo drug delivery via US with US-responsible carriers: 1) permeabilizing cellular membrane, 2) increasing the permeability of vessels, and 3) promoting cellular endocytotic uptake. We then reviewed the state-of-the-art materials for US-triggered drug delivery, including conventional US contrast agents, and nanocarrier formulations, such as inorganic nanoparticles and gas vesicles. EXPERT OPINION: In this article, we summarized recent progress for each of US-responsible drug carrier, focusing on the routes of enhancing delivery and applications. The mechanisms of interaction between US-responsible carriers and US waves, such as cavitation, streaming, hyperthermia, and ROS, as well as how those interactions can improve drug release and cell/tissue uptake.

Overcoming Hypoxia-Induced Drug Resistance via Promotion of Drug Uptake and Reoxygenation by Acousto-Mechanical Oxygen Delivery.

Hypoxia-induced drug resistance (HDR) is a critical issue in cancer therapy. The presence of hypoxic tumor cells impedes drug uptake and reduces the cytotoxicity of chemotherapeutic drugs, leading to HDR and increasing the probability of tumor recurrence and metastasis. Microbubbles, which are used as an ultrasound contrast agent and drug/gas carrier, can locally deliver drugs/gas and produce an acousto-mechanical effect to enhance cell permeability under ultrasound sonication. The present study applied oxygen-loaded microbubbles (OMBs) to evaluate the mechanisms of overcoming HDR via promotion of drug uptake and reoxygenation. A hypoxic mouse prostate tumor cell model was established by hypoxic incubation for 4 h. After OMB treatment, the permeability of HDR cells was enhanced by 23 ± 5% and doxorubicin uptake was increased by 11 ± 7%. The 61 ± 14% reoxygenation of HDR cells increased the cytotoxicity of doxorubicin from 18 ± 4% to 58 ± 6%. In combination treatment with OMB and doxorubicin, the relative contributions of uptake promotion and reoxygenation towards overcoming HDR were 11 ± 7% and 28 ± 10%, respectively. Our study demonstrated that reoxygenation of hypoxic conditions is a critical mechanism in the inhibition of HDR and enhancing the outcome of OMB treatment.

Ultrasound-activated nanomaterials for sonodynamic cancer theranostics.

Despite intensive efforts to develop diagnostic and therapeutic tools, the successful treatment of cancer is still hampered by the obscure boundary between cancerous cells and normal cells, recurrence of the cancer, and the development of drug resistance during chemotherapy. In recent years, sonodynamic therapy (SDT), employing therapeutic ultrasound with sonosensitizers, has attracted attention as a potentially promising approach for cancer therapy. This review describes the current understanding of the mechanisms and the preclinical and clinical efficacy of SDT-based applications in tumors, providing an insight into the therapeutic potential offered by SDT. The limitations and future directions of this emerging treatment are also discussed.

The Impact of Surface Drug Distribution on the Acoustic Behavior of DOX-Loaded Microbubbles.

Previous studies have reported substantial improvement of microbubble (MB)-mediated drug delivery with ultrasound when drugs are loaded onto the MB shell compared with a physical mixture. However, drug loading may affect shell properties that determine the acoustic responsiveness of MBs, producing unpredictable outcomes. The aim of this study is to reveal how the surface loaded drug (doxorubicin, DOX) affects the acoustic properties of MBs. A suitable formulation of MBs for DOX loading was first identified by regulating the proportion of two lipid materials (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phospho-rac-glycerol sodium salt (DSPG)) with distinct electrostatic properties. We found that the DOX loading capacity of MBs was determined by the proportion of DSPG, since there was an electrostatic interaction with DOX. The DOX payload reduced the lipid fluidity of MBs, although this effect was dependent on the spatial uniformity of DOX on the MB shell surface. Loading DOX onto MBs enhanced acoustic stability 1.5-fold, decreased the resonance frequency from 12-14 MHz to 5-7 MHz, and reduced stable cavitation dose by 1.5-fold, but did not affect the stable cavitation threshold (300 kPa). Our study demonstrated that the DOX reduces lipid fluidity and decreases the elasticity of the MB shell, thereby influencing the acoustic properties of MBs.

Dynamic Ultrasound Assessment of Median Nerve Mobility Changes Following Corticosteroid Injection and Carpal Tunnel Release in Patients With Carpal Tunnel Syndrome.

Decreased median nerve (MN) mobility was found in patients with carpal tunnel syndrome (CTS) and was inversely associated with symptom severity. It is unclear whether MN mobility can be restored with interventions. This study compared the changes in MN mobility and clinical outcomes after interventions. Forty-six patients with CTS received an injection (n = 23) or surgery (n = 23). Clinical outcomes [Visual Analogue Scale; Boston Carpal Tunnel Questionnaire (BCTQ), which includes the Symptom Severity Scale and Functional Status Scale; median nerve cross-sectional area; and dynamic ultrasound MN mobility parameters (amplitude, and R2 value and curvature of the fitted curves of MN transverse sliding)] were assessed at baseline and 12 weeks after the interventions. At baseline, the BCTQ-Functional Status Scale and median nerve cross-sectional area showed significant inter-treatment differences. At 12 weeks, both treatments had significant improvements in BCTQ-Symptom Severity Scale and Visual Analogue Scale scores and median nerve cross-sectional area, but with greater improvements in BCTQ-Functional Status Scale scores observed in those who received surgery than in those who received injections. MN mobility was insignificantly affected by both treatments. The additional application of dynamic ultrasound evaluation may help to discriminate the severity of CTS initially; however, its prognostic value to predict clinical outcomes after interventions in patients with CTS is limited.

Sonogenetic-Based Neuromodulation for the Amelioration of Parkinson's Disease.

Sonogenetics is a promising strategy allowing the noninvasive and selective activation of targeted neurons in deep brain regions; nevertheless, its therapeutic outcome for neurodegeneration diseases that need long-term treatment remains to be verified. We previously enhanced the ultrasound (US) sensitivity of targeted cells by genetic modification with an engineered auditory-sensing protein, mPrestin (N7T, N308S). In this study, we expressed mPrestin in the dopaminergic neurons of the substantia nigra in Parkinson's disease (PD) mice and used 0.5 MHz US for repeated and localized brain stimulation. The mPrestin expression in dopaminergic neurons persisted for at least 56 days after a single shot of adeno-associated virus, suggesting that the period of expression was long enough for US treatment in mice. Compared to untreated mice, US stimulation ameliorated the dopaminergic neurodegeneration 10-fold and mitigated the PD symptoms of the mice 4-fold, suggesting that this sonogenetic strategy has the clinical potential to treat neurodegenerative diseases.

Sonogenetic Modulation of Cellular Activities in Mammalian Cells.

Ultrasound is acoustic waves that can penetrate deeply into tissue in a focused manner with limited adverse effects on cells. As such, ultrasound has been widely used for clinical diagnosis for several decades. Ultrasound induces bioeffects in tissues, providing potential value in therapeutic applications. However, the intrinsic millimeter scale of the ultrasound focal zone represents a challenge with respect to minimizing the illuminated regions to perturb target cells in a precise manner. To control a specific cell population or even single cells, sonogenetic tools that combine ultrasound and genetic methods have been recently developed. With these approaches, several ultrasound-responsive proteins are heterologously introduced into target cells, which enhances the cells' ability to respond to ultrasound stimulation. With optimization of the ultrasound parameters, these tools can specifically manipulate activities in genetically defined cells but not in unmodified cells present in the ultrasound-illuminated regions. These approaches provide new strategies for noninvasive modulation of target cells in various therapeutic applications.

3-D Ultrafast Ultrasound Imaging of Microbubbles Trapped Using an Acoustic Vortex.

Increasing the local concentration of microbubbles (MBs) within the blood flow plays a crucial role in several medical applications, but there are few imaging modalities available for volumetric tracking of the aggregated MBs in real time. Here we describe a device integrating acoustic vortex tweezers (AVTs) and ultrasound plane-wave imaging (PWI) to achieve the goal of controlling the spatial distribution of MBs in blood vessels and simultaneously monitoring this process using the same probe. Experiments were conducted using a 5-MHz 2-D array ultrasound probe (with three cycles of excitation at an acoustic pressure of 2000 kPa) and 1.2- [Formula: see text]-diameter MBs at a flow rate of 20 mm/s. The AVT waveform was produced by modulating the repetition frequency of the transmitted pulse asymmetrically (4 and 8 kHz at the inflow and outflow ends, respectively). In order to simultaneously capture MBs and carry out imaging with the same probe, the asymmetric AVT pulse signal and the ultrasound-imaging pulse signal were arranged in a staggered series, and the imaging was carried out using plane-wave pulses at nine angles (-7° to 7°) in compounded PWI (volume rate: 200 Hz). Microscopy observations showed that freely suspended MBs could indeed be gathered by the asymmetric AVT in the flow field to form an MBs cluster with a spot size of about [Formula: see text], which could resist the flow to remain at a fixed location for about 22 s. After the asymmetric AVT signal and the ultrasound-imaging pulse signal were turned on for 1 s, the ultrasound 3-D image showed that the signal intensity of the MB clusters increased by 13.1 dB ± 2.9 dB in relation to the background area. These results show that the proposed strategy can be used to accumulate flowing MBs at a desired location and to simultaneously observe this phenomenon. This tool could be used in the future to improve the outcomes of MB-related treatments for various diseases.