Search

Objective: Subharmonic Aided Pressure Estimation (SHAPE) is a noninvasive technique for estimating organ-level blood pressure using the strong correlation between the subharmonic signal and ambient pressure. The compressible gas core of microbubbles enables them to generate linear and nonlinear acoustic responses when exposed to ultrasound. Here, the sulfur hexafluoride (SF 6 ) gas core of SonoVue (known as Lumason in the United States), a clinical contrast agent, was exchanged with a perfluorobutane (PFB) core to investigate its effect on the SHAPE response., Methods: Excitations of 25-700 kPa peak negative pressure (PNP) and 3 MHz transmission frequency were used to study in vitro the effects of overpressure changes ranging from 5 to 25 kPa (37-186 mm Hg)., Results: Unlike SonoVue with SF 6 , at low PNPs (<400 kPa), SonoVue with a PFB gas core exhibited no subharmonic at the atmospheric pressure, but during pressurization, a stable subharmonic response (maximum of 25 dB at 100 kPa PNP and 20 kPa Overpressure) appeared. SonoVue with a PFB gas core showed an increase in subharmonics with overpressure at high PNPs (>400 kPa), which was not observed before in normal SonoVue or other lipid microbubbles. With negligible size distribution difference between these two microbubbles, these effects on subharmonic generation are likely due to the gas core, casting new light on the mechanism by which ambient overpressure affects subharmonic., Conclusion: This study may inform future SHAPE technique developments., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Perfluoropropane droplets (PD) are nanometer-sized particles that can be formulated from commercially available contrast agents. The preferential retention of PDs in diseased microvascular beds can be detected by ultrasound imaging techniques after acoustic activation and offers an opportunity for the detection of such processes as scar formation or inflammation. We hypothesized that in the presence of ischemia/reperfusion (I/R) injury, retention of intravenously injected PDs would be enhanced., Methods: Using an established intravital microscopy model of rat cremaster microcirculation, we determined the retention and subsequent acoustic activation behavior of PDs in exteriorized rat cremaster tissue. DiI-labeled droplets (200 µL) were administered intravenously. Acoustic activation was achieved with a clinical ultrasound system at two ultrasound frequencies (1.5 and 7 MHz)., Results: Fluorescent microbubbles could be detected in the microvasculature after intravenous injection of PDs and subsequent acoustic activation. Increased retention of PDs was observed in the I/R group compared with control group with both ultrasound frequencies (p < 0.05). Using higher-resolution microscopy, we found evidence that some droplets extravasate to the outside of the endothelial border or are potentially engulfed by leukocytes., Conclusion: Our data indicate that targeted imaging of the developing scar zones might be possible with ultrasound activation of intravenously injected PDs, and a method of targeting therapies to these same regions could be developed., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2024 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Both microbubble ultrasound contrast agents and acoustic phase change droplets (APCD) have been explored in hepatocellular carcinoma (HCC). This work aimed to evaluate changes to the HCC microenvironment following either microbubble or APCD destruction in a syngeneic pre-clinical model., Methods: Mouse RIL-175 HCC tumors were grown in the right flank of 64 immunocompetent mice. Pre-treatment, photoacoustic volumetric tumor oxygenation, and power Doppler measurements were obtained using a Vevo 3100 system (VisualSonics, Toronto, Canada). The experimental groups received a 0.1 mL bolus injection of either Definity ultrasound contrast agent (Lantheus Medical Imaging) or APCD fabricated by condensing Definity. Following injection, ultrasound destruction was performed using flash-replenishment sequences on a Sequoia with a 10L4 probe (Siemens) for the duration of enhancement. Tumor oxygenation and power Doppler measurements were then repeated immediately post-ultrasound treatment. Twenty-four hours post-treatment, animals were euthanized, and tumors were harvested and stained for CD31, Cleaved Caspase 3 and CD45., Results: Imaging biomarkers demonstrated a significant reduction in percent vascularity following either microbubble or APCD destruction in the tumor microenvironment ( p < 0.022) but no significant changes in tumor oxygenation (p = 0.12). Similarly, immunohistochemistry data demonstrated a significant decrease in CD31 expression (p < 0.042) and an increase in apoptosis (p < 0.014) in tumors treated with destroyed microbubbles or APCD relative to controls. Finally, a significant increase in CD45 expression was observed in tumors treated with APCD (p = 0.046), indicating an increase in tumor immune response., Conclusion: Ultrasound-triggered destruction of both microbubbles and APCD reduces vascularity, increases apoptosis, and may also increase immune response in this HCC model., Competing Interests: Conflict of Interest CEW: Consultant for Bracco Diagnostics, speaking bureau for Canon Medical Systems USA, consultant for SonoSim. JRE: Equipment, drug, grant support and honorarium from GE Healthcare. Drug support from Bracco. Drug, grant support and honorarium from Lantheus Medical Imaging. Equipment support from Siemens. Consultant for SonoSim. Royalties from Elsevier. HF, QL, MAW, JBL: None., (Copyright © 2024 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Endobronchial ultrasound (EBUS) is commonly used to guide transbronchial needle biopsies for the staging of lymph nodes in non-small cell lung cancer patients. Although contrast-enhanced ultrasound (CEUS) and microbubbles (MBs) can improve the diagnostic accuracy in tumors, the ability of contrast-enhanced EBUS (CE-EBUS) to image MBs has not yet been comprehensively evaluated. In this study, we assessed the ability of a CE-EBUS system (Olympus EU-ME2 PREMIER and BF-UC180F bronchoscope) to detect laboratory-synthesized MBs in comparison to clinical (Toshiba Aplio SSA-790A) and pre-clinical (VisualSonics Vevo 2100) CEUS systems in vitro and in vivo, respectively., Methods: Agar flow phantoms and reference tissue were used to assess CE-EBUS MB imaging in vitro, and A549 tumor-bearing athymic nude and AE17-OVA tumor-bearing C57BL/6 mice were used to assess MB detectability and perfusion in vivo, respectively., Results: Results revealed that despite the lower sensitivity of CE-EBUS to MB concentration in comparison to clinical CEUS, CE-EBUS yielded a similar contrast-to-tissue ratio (CTR) in vitro of 28.9 ± 4.5 dB for CE-EBUS, compared with 29.7 ± 2.6 dB for clinical CEUS (p < 0.05). In vivo, CE-EBUS generated a perfusion curve highly correlated with that obtained with the pre-clinical CEUS system (Pearson correlation coefficient = 0.927, p < 0.05). Moreover, CE-EBUS yielded a CTR 2.7 times higher than that obtained with the pre-clinical ultrasound system., Conclusion: These findings together suggest that CE-EBUS can perform contrast imaging comparable to that produced by commercial pre-clinical and clinical ultrasound systems, with potential for clinical characterization of mediastinal lymph nodes in lung cancer patients., Competing Interests: Conflict of interest K.Y. is a consultant for Olympus, and the work was partially supported by a research grant from Olympus Corporation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The objective of this work was to study microbubble-enhanced temperature elevation with high-intensity focused ultrasound (HIFU) at different acoustic pressures and under image guidance. The microbubbles were administered with either local or vascular injections (that mimic systemic injections) in perfused and non-perfused ex vivo porcine liver under ultrasound image guidance., Methods: Porcine liver was insonified for 30 s with a single-element HIFU transducer (0.9 MHz, 0.413 ms, 82% duty cycle, focal pressures of 0.6-3.5 MPa). Contrast microbubbles were injected either locally or through the vasculature. A needle thermocouple at the focus measured temperature elevation. Diagnostic ultrasound (Philips iU22, C5-1 probe) guided placement of the thermocouple and delivery of microbubbles and monitored the procedure in real time., Results: At lower acoustic pressures (0.6 and 1.2 MPa) in non-perfused liver, inertial cavitation of the injected microbubbles led to greater temperatures at the focus compared with HIFU-only treatments. At higher pressures (2.4 and 3.5 MPa) native inertial cavitation in the tissue (without injecting microbubbles) resulted in temperature elevations similar to those after injecting microbubbles. The heated area was larger when using microbubbles at all pressures. In the presence of perfusion, only local injections provided a sufficiently high concentration of microbubbles necessary for significant temperature enhancement., Conclusion: Local injections of microbubbles provide a higher concentration of microbubbles in a smaller area, avoiding acoustic shadowing, and can lead to higher temperature elevation at lower pressures and increase the size of the heated area at all pressures., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: The sensitivity of the acoustic response of microbubbles, specifically a strong correlation between their subharmonic response and the ambient pressure, has motivated development of a non-invasive subharmonic-aided pressure estimation (SHAPE) method. However, this correlation has previously been found to vary depending on the microbubble type, the acoustic excitation and the hydrostatic pressure range. In this study, the ambient pressure sensitivity of microbubble response was investigated., Methods: The fundamental, subharmonic, second harmonic and ultraharmonic responses from an in-house lipid-coated microbubble were measured for excitations with peak negative pressures (PNPs) of 50-700 kPa and frequencies of 2, 3 and 4 MHz in the ambient overpressure range 0-25 kPa (0-187 mmHg) in an in vitro setup., Results: The subharmonic response typically has three stages-occurrence, growth and saturation-with increasing excitation PNP. We find distinct decreasing and increasing variations of the subharmonic signal with overpressure that are closely related to the threshold of subharmonic generation in a lipid-shelled microbubble. Above the excitation threshold, that is, in the growth-saturation phase, subharmonic signals decreased linearly with slopes as high as -0.56 dB/kPa with ambient pressure increase; below the threshold excitation (at atmospheric pressure), increasing overpressure triggers subharmonic generation, indicating a lowering of subharmonic threshold, and therefore leads to an increase in subharmonic with overpressure, the maximum enhancement being ∼11 dB for 15 kPa overpressure at 2 MHz and 100 kPa PNP., Conclusion: This study indicates the possible development of novel and improved SHAPE methodologies., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Objective: Currently available cytotoxic treatments have limited effect on pancreatic ductal adenocarcinoma (PDAC) because desmoplastic stroma limits drug delivery. Efforts have been made to overcome these barriers by drug targeting the tumor microenvironment. Results so far are promising, but without clinical impact. Our aim was to investigate whether ultrasound and microbubbles could improve the uptake and therapeutic response of conventional chemotherapy., Methods: Orthotopic pancreatic tumors growing in mice were treated with commercially available FOLFIRINOX (fluorouracil, irinotecan, oxaliplatin and calcium folinate) and SonoVue microbubbles combined with focused ultrasound. Tumor uptake of platinum (Pt) was measured by inductively coupled plasma mass spectroscopy (ICP-MS), and tumor volumes were measured by ultrasound imaging., Discussion: Uptake of Pt, the active ingredient of oxaliplatin, was significantly increased after ultrasound treatment of orthotopic PDAC tumors. Multiple injections with FOLFIRONOX increased the amount of Pt in tumors. However, the enhanced accumulation did not improve therapeutic response. Increased uptake of Pt confirms that ultrasound and microbubbles have potential in clinical practice with existing drugs., Conclusion: The lack of therapeutic response, despite increased uptake in tumor tissue, emphasizes the importance of studying how to overcome stromal barriers., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC)-mediated therapies have been found to restore perfusion and enhance drug/gene delivery. Because of the potentially longer circulation time and relative ease of storage and reconstitution of polymer-shelled MBs compared with lipid MBs, we investigated the dynamic behavior of polymer microbubbles and their therapeutic potential for sonoreperfusion (SRP) therapy., Methods: The fate of polymer MBs during a single long tone-burst exposure (1 MHz, 5 ms) at various acoustic pressures and MB concentrations was recorded via high-speed microscopy and passive cavitation detection (PCD). SRP efficacy of the polymer MBs was investigated in an in vitro flow system and compared with that of lipid MBs., Discussion: Microscopy videos indicated that polymer MBs formed gas-filled clusters that continued to oscillate, fragment and form new gas-filled clusters during the single US burst. PCD confirmed continued acoustic activity throughout the 5-ms US excitation. SRP efficacy with polymer MBs increased with pulse duration and acoustic pressure similarly to that with lipid MBs but no significant differences were found between polymer and lipid MBs., Conclusion: These data suggest that persistent cavitation activity from polymer MBs during long tone-burst US excitation confers excellent reperfusion efficacy., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments., Competing Interests: Conflict of interest disclosure The authors declare no known conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ultrasound has important applications, predominantly in the field of diagnostic imaging. Presently, colloidal systems such as microbubbles, phase-change emulsion droplets and particle systems with acoustic properties and multiresponsiveness are being developed to address typical issues faced when using commercial ultrasound contrast agents, and to extend the utility of such systems to targeted drug delivery and multimodal imaging. Current technologies and increasing research data on the chemistry, physics and materials science of new colloidal systems are also leading to the development of more complex, novel and application-specific colloidal assemblies with ultrasound contrast enhancement and other properties, which could be beneficial for multiple biomedical applications, especially imaging-guided treatments. In this article, we review recent developments in new colloids with applications that use ultrasound contrast enhancement. This work also highlights the emergence of colloidal materials fabricated from or modified with biologically derived and bio-inspired materials, particularly in the form of biopolymers and biomembranes. Challenges, limitations, potential developments and future directions of these next-generation colloidal systems are also presented and discussed., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Contrast-enhanced ultrasound (CEUS) is clinically used to image the microcirculation at lower imaging frequencies (<2 MHz). Recently, plane-wave acquisitions and Doppler processing have revealed improved microbubble sensitivity, enabling CEUS use at higher frequencies (15 MHz) and the ability to image simultaneously blood flow in the micro- and macrocirculations. We used this approach to assess acute and chronic blood flow changes within contused spinal cord in a rodent spinal cord injury model. Immediately after spinal cord injury, we found significant differences in perfusion deficit between moderate and severe injuries (1.73 ± 0.1 mm 2 vs. 3.2 ± 0.3 mm 2 , respectively), as well as a delay in microbubble arrival time in tissue adjacent to the injury site (0.97 ± 0.1 s vs. 1.54 ± 0.1 s, respectively). Acutely, morphological changes to central sulcal arteries were observed where vessels rostral to the contusion were displaced 4.8 ± 2.2° and 8.2 ± 3.1° anteriorly, and vessels caudal to the contusion 17.8 ± 3.9° and 24.2 ± 4.1° posteriorly, respectively, for moderate and severe injuries. Significant correlation of the acute perfusion deficit and arrival time were found with the chronic assessment of locomotive function and histological estimate of spared spinal cord tissue., (Copyright © 2022 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

We present a computational study of the behaviour of a lipid-coated SonoVue microbubble with initial radius 1 µm ≤ R 0 ≤ 2 µm, excited at frequencies (200-1500 kHz) significantly below the linear resonance frequency and pressure amplitudes of up to 1500 kPa-an excitation regime used in many applications of focused ultrasound. The bubble dynamics are simulated using the Rayleigh-Plesset equation and the Gilmore equation, in conjunction with the Marmottant model for the lipid monolayer coating. Also, a new continuously differentiable variant of the Marmottant model is introduced. Below the onset of inertial cavitation, a linear regime is identified in which the maximum pressure at the bubble wall is linearly proportional to the excitation pressure amplitude and the mechanical index. This linear regime is bounded by the Blake pressure, and, in line with recent in vitro experiments, the onset of inertial cavitation is found to occur at an excitation pressure amplitude of approximately 130-190 kPa, depending on the initial bubble size. In the nonlinear regime the maximum pressure at the bubble wall is found to be readily predicted by the maximum bubble radius, and both the Rayleigh-Plesset and Gilmore equations are shown to predict the onset of sub- and ultraharmonic frequencies of the acoustic emissions compared with in vitro experiments. Neither the surface dilational viscosity of the lipid monolayer nor the compressibility of the liquid has a discernible influence on the quantities studied, but accounting for the lipid coating is critical for accurate prediction of the bubble behaviour. The Gilmore equation is shown to be valid for the bubbles and excitation regime considered, and the Rayleigh-Plesset equation also provides accurate qualitative predictions, even though it is outside its range of validity for many of the cases considered., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

High-intensity focused ultrasound (HIFU) is a non-invasive tool that can be used for targeted thermal ablation treatments. Currently, HIFU is clinically approved for treatment of uterine fibroids, various cancers, and certain brain applications. However, for brain applications such as essential tremors, HIFU can only be used to treat limited areas confined to the center of the brain because of geometrical limitations (shape of the transducer and skull). A major obstacle to advancing this technology is the inability to treat non-central brain locations without causing damage to the skin and/or skull. Previous research has indicated that cavitation-induced bubbles or microbubble contrast agents can be used to enhance HIFU treatments by increasing ablation regions and shortening acoustic exposures at lower acoustic pressures. However, little research has been done to explore the interplay between microbubble concentration and pressure amplitude on HIFU treatments. We developed an in vitro experimental setup to study lesion formation at three different acoustic pressures and three microbubble concentrations. Real-time ultrasound imaging was integrated to monitor initial microbubble concentration and subsequent behavior during the HIFU treatments. Depending on the pressure used for the HIFU treatment, there was an optimal concentration of microbubbles that led to enhanced heating in the focal area. If the concentration of microbubbles was too high, the treatment was detrimentally affected because of non-linear attenuation by the pre-focal microbubbles. Additionally, the real-time ultrasound imaging provided a reliable method to monitor microbubble activity during the HIFU treatments, which is important for translation to in vivo HIFU applications with microbubbles., (Copyright © 2021 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Intra-operative contrast-enhanced ultrasound (CEUS) is a relatively standardized procedure in brain neurosurgery, but it is still underused in spinal cord and intramedullary tumor evaluation. We reviewed and analyzed the intra-operative data from a surgical series of patients harboring intramedullary spinal cord tumors who underwent surgery under CEUS guidance. CEUS was performed in 12 patients (age range: 13-55 y); all lesions had ill-defined boundaries or peritumoral cysts at preliminary intra-operative B-mode ultrasound. CEUS highlighted the tumors in all cases. The contrast agent's spinal distribution revealed different phases (arterial, peak, washout), as observed in the brain, but these appeared to be slower and less intense. In our experience, intra-operative CEUS allows surgeons to assess spinal cord perfusion and highlight intramedullary tumors in real time. As for other imaging modalities, ultrasound contrast agents add valuable information over baseline imaging, and their use should be fostered to better understand microbubble distribution dynamics., Competing Interests: Conflict of interest disclosure All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this article., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Mechanical effects of microbubbles on tissues are central to many emerging ultrasound applications. Here, we investigated the acoustic radiation force a microbubble exerts on tissue at clinically relevant therapeutic ultrasound parameters. Individual microbubbles administered into a wall-less hydrogel channel (diameter: 25-100 µm, Young's modulus: 2-8.7 kPa) were exposed to an acoustic pulse (centre frequency: 1 MHz, pulse length: 10 ms, peak-rarefactional pressures: 0.6-1.0 MPa). Using high-speed microscopy, each microbubble was tracked as it pushed against the hydrogel wall. We found that a single microbubble can transiently deform a soft tissue-mimicking material by several micrometres, producing tissue loading-unloading curves that were similar to those produced using other indentation-based methods. Indentation depths were linked to gel stiffness. Using a mathematical model fitted to the deformation curves, we estimated the radiation force on each bubble (typically tens of nanonewtons) and the viscosity of the gels. These results provide insight into the forces exerted on tissues during ultrasound therapy and indicate a potential source of bio-effects., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Recent advances in the field of monodisperse microbubble synthesis by flow focusing allow for the production of foam-free, highly concentrated and monodisperse lipid-coated microbubble suspensions. It has been found that in vitro, such monodisperse ultrasound contrast agents (UCAs) improve the sensitivity of contrast-enhanced ultrasound imaging. Here, we present the first in vivo study in the left ventricle of rat and pig with this new monodisperse bubble agent. We systematically characterize the acoustic sensitivity and safety of the agent at an imaging frequency of 2.5 MHz as compared with three commercial polydisperse UCAs (SonoVue/Lumason, Definity/Luminity and Optison) and one research-grade polydisperse agent with the same shell composition as the monodisperse bubbles. The monodisperse microbubbles, which had a diameter of 4.2 μm, crossed the pulmonary vasculature, and their echo signal could be measured at least as long as that of the polydisperse UCAs, indicating that microfluidically formed monodisperse microbubbles are stable in vivo. Furthermore, it was found that the sensitivity of the monodisperse agent, expressed as the mean echo power per injected bubble, was at least 10 times higher than that of the polydisperse UCAs. Finally, the safety profile of the monodisperse microbubble suspension was evaluated by injecting 400 and 2000 times the imaging dose, and neither physiologic nor pathologic changes were found, which is a first indication that monodisperse lipid-coated microbubbles formed by flow focusing are safe for in vivo use. The more uniform acoustic response and corresponding increased imaging sensitivity of the monodisperse agent may boost emerging applications of microbubbles and ultrasound such as molecular imaging and therapy., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Submicron phase-change droplets are an emerging class of ultrasound contrast agent. Compared with microbubbles, their relatively small size and increased stability offer the potential to passively extravasate and accumulate in solid tumors through the enhanced permeability and retention effect. Under exposure to sufficiently powerful ultrasound, these droplets can convert into in situ gas microbubbles and thus be used as an extravascular-specific contrast agent. However, in vivo imaging methods to detect extravasated droplets have yet to be established. Here, we develop an ultrasound imaging pulse sequence within diagnostic safety limits to selectively detect droplet extravasation in tumors. Tumor-bearing mice were injected with submicron perfluorobutane droplets and interrogated with our imaging-vaporization-imaging sequence. By use of a pulse subtraction method, median droplet extravasation signal relative to the total signal within the tumor was estimated to be E tumor =37±5% compared with the kidney E kidney =-2±8% (p < 0.001). This work contributes toward the advancement of volatile phase-shift droplets as a next-generation ultrasound agent for imaging and therapy., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Ultrasound is extensively used in medical imaging, being safe and inexpensive and operating in real time. Its scope of applications has been widely broadened by the use of ultrasound contrast agents (UCAs) in the form of microscopic bubbles coated by a biocompatible shell. Their increased use has motivated a large amount of research to understand and characterize their physical properties as well as their interaction with the ultrasound field and their surrounding environment. Here we review the theoretical models that have been proposed to study and predict the behavior of UCAs. We begin with a brief introduction on the development of UCAs. We then present the basics of free-gas-bubble dynamics upon which UCA modeling is based. We review extensively the linear and non-linear models for shell elasticity and viscosity and present models for non-spherical and asymmetric bubble oscillations, especially in the presence of surrounding walls or tissue. Then, higher-order effects such as microstreaming, shedding and acoustic radiation forces are considered. We conclude this review with promising directions for the modeling and development of novel agents., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Microbubble ultrasound contrast agents have now been in use for several decades and their safety and efficacy in a wide range of diagnostic applications have been well established. Recent progress in imaging technology is facilitating exciting developments in techniques such as molecular, 3-D and super resolution imaging and new agents are now being developed to meet their specific requirements. In parallel, there have been significant advances in the therapeutic applications of microbubbles, with recent clinical trials demonstrating drug delivery across the blood-brain barrier and into solid tumours. New agents are similarly being tailored toward these applications, including nanoscale microbubble precursors offering superior circulation times and tissue penetration. The development of novel agents does, however, present several challenges, particularly regarding the regulatory framework. This article reviews the developments in agents for diagnostic, therapeutic and "theranostic" applications; novel manufacturing techniques; and the opportunities and challenges for their commercial and clinical translation., Competing Interests: Declaration of Competing Interest TB and SC are full time employees of Bracco Suisse. The other authors are hold full time academic positions and have the following company affiliations: ES is a founder and non-executive director of SonoTarg Ltd.; MB is a founder and chief scientific officer of Respirogen Inc., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

This World Federation for Ultrasound in Medicine and Biology position paper reviews the diagnostic potential of ultrasound contrast agents for clinical decision-making and provides general advice for optimal contrast-enhanced ultrasound performance in musculoskeletal issues. In this domain, contrast-enhanced ultrasound performance has increasingly been investigated with promising results, but still lacks everyday clinical application and standardized techniques; therefore, experts summarized current knowledge according to published evidence and best personal experience. The goal was to intensify and standardize the use and administration of ultrasound contrast agents to facilitate correct diagnoses and ultimately to improve the management and outcomes of patients., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The epidemic of increasing fatty liver disease and liver cancer worldwide, and especially in Western society, has given new importance to non-invasive liver imaging. Contrast-enhanced ultrasound (CEUS) using microbubble contrast agents provides unique advantages over computed tomography (CT) and magnetic resonance imaging (MRI), the currently established methods. CEUS provides determination of malignancy and allows excellent differential diagnosis of a focal liver mass, based on arterial phase enhancement patterns and assessment of the timing and intensity of washout. Today, increased use of CEUS has provided safe and rapid diagnosis of incidentally detected liver masses, improved multidisciplinary management of nodules in a cirrhotic liver, facilitated ablative therapy for liver tumors and allowed diagnosis of hepatocellular carcinoma without biopsy. Benefits of CEUS include the dynamic real-time depiction of tumor perfusion and the fact that it is a purely intravascular agent, accurately reflecting tumoral and inflammatory blood flow. CEUS has many similarities to contrast-enhanced CT and MRI but also unique differences, which are described. The integration of CEUS into a multimodality imaging setting optimizes patient care., Competing Interests: Conflict of interest disclosure The authors declare no competing interests., (Copyright © 2020 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Ultrasound contrast agents (UCAs) have opened up immense diagnostic possibilities by combined use of indicator dilution principles and dynamic contrast-enhanced ultrasound (DCE-US) imaging. UCAs are microbubbles encapsulated in a biocompatible shell. With a rheology comparable to that of red blood cells, UCAs provide an intravascular indicator for functional imaging of the (micro)vasculature by quantitative DCE-US. Several models of the UCA intravascular kinetics have been proposed to provide functional quantitative maps, aiding diagnosis of different pathological conditions. This article is a comprehensive review of the available methods for quantitative DCE-US imaging based on temporal, spatial and spatiotemporal analysis of the UCA kinetics. The recent introduction of novel UCAs that are targeted to specific vascular receptors has advanced DCE-US to a molecular imaging modality. In parallel, new kinetic models of increased complexity have been developed. The extraction of multiple quantitative maps, reflecting complementary variables of the underlying physiological processes, requires an integrative approach to their interpretation. A probabilistic framework based on emerging machine-learning methods represents nowadays the ultimate approach, improving the diagnostic accuracy of DCE-US imaging by optimal combination of the extracted complementary information. The current value and future perspective of all these advances are critically discussed., (Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Controlling microbubble-mediated drug delivery requires the underlying biological and physical mechanisms to be unraveled. To image both microbubble oscillation upon ultrasound insonification and the resulting cellular response, we developed an optical imaging system that can achieve the necessary nanosecond temporal and nanometer spatial resolutions. We coupled the Brandaris 128 ultra-high-speed camera (up to 25 million frames per second) to a custom-built Nikon A1R+ confocal microscope. The unique capabilities of this combined system are demonstrated with three experiments showing microbubble oscillation leading to either endothelial drug delivery, bacterial biofilm disruption, or structural changes in the microbubble coating. In conclusion, using this state-of-the-art optical imaging system, microbubble-mediated drug delivery can be studied with high temporal resolution to resolve microbubble oscillation and high spatial resolution and detector sensitivity to discern cellular response. Combining these two imaging technologies will substantially advance our knowledge on microbubble behavior and its role in drug delivery., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Dynamic contrast-enhanced ultrasound (DCE-US) has been proposed as a powerful tool for cancer diagnosis by estimation of perfusion and dispersion parameters reflecting angiogenic vascular changes. This work was aimed at identifying which vascular features are reflected by the estimated perfusion and dispersion parameters through comparison with acoustic angiography (AA). AA is a high-resolution technique that allows quantification of vascular morphology. Three-dimensional AA and 2-D DCE-US bolus acquisitions were used to monitor the growth of fibrosarcoma tumors in nine rats. AA-derived vascular properties were analyzed along with DCE-US perfusion and dispersion to investigate the differences between tumor and control and their evolution in time. AA-derived microvascular density and DCE-US perfusion exhibited good agreement, confirmed by their spatial distributions. No vascular feature was correlated with dispersion. Yet, dispersion provided better cancer classification than perfusion. We therefore hypothesize that dispersion characterizes vessels that are smaller than those visible with AA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Phase-shift droplets can be converted by sound from low-echogenicity, liquid-core agents into highly echogenic microbubbles. Many proposed applications in imaging and therapy take advantage of the high spatiotemporal control over this dynamic transition. Although some studies have reported increased circulation time of the droplets compared with microbubbles, few have directly explored the impact of encapsulation on droplet performance. With the goal of developing nanoscale droplets with increased circulatory persistence, we first evaluate the half-life of several candidate phospholipid encapsulations in vitro at clinical frequencies. To evaluate in vivo circulatory persistence, we develop a technique to periodically measure droplet vaporization from high-frequency B-mode scans of a mouse kidney. Results show that longer acyl chain phospholipids can dramatically reduce droplet degradation, increasing median half-life in vitro to 25.6 min-a 50-fold increase over droplets formed from phospholipids commonly used for clinical microbubbles. In vivo, the best-performing droplet formulations showed a median half-life of 18.4 min, more than a 35-fold increase in circulatory half-life compared with microbubbles with the same encapsulation in vivo. These findings also point to possible refinements that may improve nanoscale phase-shift droplet performance beyond those measured here., (Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

Contrast-enhanced ultrasound (CEUS) is an innovative ultrasound technique capable of visualizing both the macro- and microvasculature of tissues. In this prospective pilot study, we evaluated the feasibility of using CEUS to visualize the microvasculature of uterine fibroids and compared CEUS with conventional ultrasound. Four women with fibroids underwent gray-scale ultrasound, sonoelastography and power/color Doppler scans followed by CEUS examination. Analysis of CEUS images revealed initial perfusion of the peripheral rim, that is, a pseudo-capsule, followed by enhancement of the entire lesion through vessels traveling from the exterior to the interior of the fibroid. The pseudo-capsules exhibited slight hyper-enhancement, making a clear delineation of the fibroids possible. The centers of three fibroids exhibited areas lacking vascularization, information not obtainable with the other imaging techniques. CEUS is a feasible technique for imaging and quantifying the microvasculature of fibroids. In comparison with conventional ultrasound imaging modalities, CEUS can provide additional diagnostic information based on the microvasculature., (Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

It has been proposed that monodisperse microbubble ultrasound contrast agents further increase the signal-to-noise ratio of contrast-enhanced ultrasound imaging. Here, the sensitivity of a polydisperse pre-clinical agent was compared experimentally with that of its size- and acoustically sorted derivatives by using narrowband pressure- and frequency-dependent scattering and attenuation measurements. The sorted monodisperse agents had up to a two-orders-of-magnitude increase in sensitivity, that is, in the average scattering cross section per bubble. Moreover, we found, for the first time, that the highly non-linear response of acoustically sorted microbubbles can be exploited to confine scattering and attenuation to the focal region of ultrasound fields used in clinical imaging. This property is a result of minimal pre-focal scattering and attenuation and can be used to minimize shadowing effects in deep tissue imaging. Moreover, it potentially allows for more localized therapy using microbubbles through the spatial control of resonant microbubble oscillations., (Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

Physical characterization of an ultrasound contrast agent (UCA) aids in its safe and effective use in diagnostic and therapeutic applications. The goal of this study was to investigate the impact of temperature on the size distribution, shell properties, and stability of Definity ® , a U.S. Food and Drug Administration-approved UCA used for left ventricular opacification. A Coulter counter was modified to enable particle size measurements at physiologic temperatures. The broadband acoustic attenuation spectrum and size distribution of Definity ® were measured at room temperature (25 °C) and physiologic temperature (37 °C) and were used to estimate the viscoelastic shell properties of the agent at both temperatures. Attenuation and size distribution was measured over time to assess the effect of temperature on the temporal stability of Definity ® . The attenuation coefficient of Definity ® at 37 °C was as much as 5 dB higher than the attenuation coefficient measured at 25 °C. However, the size distributions of Definity ® at 25 °C and 37 °C were similar. The estimated shell stiffness and viscosity decreased from 1.76 ± 0.18 N/m and 0.21 × 10 -6  ± 0.07 × 10 -6 kg/s at 25 °C to 1.01 ± 0.07 N/m and 0.04 × 10 -6  ± 0.04 × 10 -6 kg/s at 37 °C, respectively. Size-dependent differences in dissolution rates were observed within the UCA population at both 25 °C and 37 °C. Additionally, cooling the diluted UCA suspension from 37 °C to 25 °C accelerated the dissolution rate. These results indicate that although temperature affects the shell properties of Definity ® and can influence the stability of Definity ® , the size distribution of this agent is not affected by a temperature increase from 25 °C to 37 °C., (Copyright © 2018 World Federation for Ultrasound in Medicine and Biology. Published by Elsevier Inc. All rights reserved.)

Contrast-enhanced intra-vascular ultrasound (CE-IVUS) imaging could provide clinicians a valuable tool to assess cardiovascular risk and guide the choice of therapeutic strategies. In this technical note, we evaluated the feasibility of combining subharmonic and ultraharmonic imaging to improve the performance of CE-IVUS. Vessel phantoms perfused with phospholipid-shelled ultrasound contrast agents were visualized using subharmonic, ultraharmonic and combined CE-IVUS modes with commercial peripheral and coronary imaging catheters. Flow channels as small as 0.8 mm and 0.5 mm were imaged at 12-MHz and 30-MHz transmit frequencies, respectively. Subharmonic and ultraharmonic imaging modes achieved a contrast-to-tissue ratio (CTR) up to 18.1 ± 1.8 dB and 19.6 ± 1.9 dB at 12-MHz, and 8.8 ± 1.8 and 12.5 ± 1.1 dB at 30-MHz transmit frequencies, respectively. Combining these modes improved the CTR to 32.5 ± 3.0 dB and 25.0 ± 1.6 dB at 12-MHz and 30-MHz transmit frequencies. These results underscore the potential of combined-mode CE-IVUS imaging. Furthermore, the demonstration of this approach with commercial catheters may serve as a first step toward the clinical translation of CE-IVUS., (Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

This study evaluated the feasibility of employing non-invasive intra-cardiac pressure estimation using subharmonic signals from ultrasound contrast agents in humans. This institutional review board-approved proof-of-concept study included 15 consenting patients scheduled for left and right heart catheterization. During the catheterization procedure, Definity was infused intra-venously at 4-10 mL/min. Ultrasound scanning was performed with a Sonix RP using pulse inversion, three incident acoustic output levels and 2.5-MHz transmit frequency. Radiofrequency data were processed and subharmonic amplitudes were compared with the pressure catheter data. The correlation coefficient between subharmonic signals and pressure catheter data ranged from -0.3 to -0.9. For acquisitions with optimum acoustic output, pressure errors between the subharmonic technique and catheter were as low as 2.6 mmHg. However, automatically determining optimum acoustic output during scanning for each patient remains to be addressed before clinical applicability can be decided., (Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

We have previously reported that long-tone-burst, high-mechanical-index ultrasound (US) and microbubble (MB) therapy can restore perfusion in both in vitro and in vivo models of microvascular obstruction (MVO). Addition of MBs to US has been found to potentiate the efficacy of thrombolytics on large venous thrombi; however, the optimal US parameters for achieving microvascular reperfusion of MVO caused by microthrombi, when combined with tissue plasminogen activator (tPA), are unknown. We sought to elucidate the specific effects of US, with and without tPA, for effective reperfusion of MVO in an in vitro model using both venous and arterial microthrombi. Venous- and arterial-type microthrombi were infused onto a mesh with 40-μm pores to simulate MVO. Pulsed US (1 MHz) was delivered with inertial cavitation (IC) (1.0 MPa, 1000 cycles, 0.33 Hz) and stable cavitation (SC) US (0.23 MPa, 20% duty cycle, 0.33 Hz) regimes while MB suspension (2 × 10 6  MBs/mL) was infused. The efficacy of sonoreperfusion with these parameters was tested with and without tPA. Sonoreperfusion efficacy was significantly greater for IC + tPA compared with tPA alone, IC, SC and SC + tPA, suggesting lytic synergism between tPA and US for both venous- and arterial-type microthrombi. In contrast to our previous in vitro studies using 1.5 MPa at 5000 US cycles without tPA, the IC regime employed herein used 90% less US energy. These findings suggest an IC regime can be used with tPA synergistically to achieve a high degree of fibrinolysis for both thrombus types., (Copyright © 2017 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Subharmonic signals backscattered from gas-filled lipid-shelled microbubbles have generated significant research interest because they can improve the detection and sensitivity of contrast-enhanced ultrasound imaging. However, the emission of subharmonic signals is strongly characterized by a temporal dependence, the origins of which have not been sufficiently elucidated. The features that influence subharmonic emissions need to be identified not only to better develop next-generation microbubble contrast agents, but also to develop more efficient subharmonic imaging (SHI) modes and therapeutic strategies. We examined the effect of microbubble filling gas on subharmonic emissions. Phospholipid shelled-microbubbles with different gaseous compositions such as sulfur hexafluoride (SF 6 ), octafluoropropane (C 3 F 8 ) or decafluorobutane (C 4 F 10 ), nitrogen (N 2 )/C 4 F 10 or air were insonated using a driving frequency of 10 MHz and peak negative pressure of 450 kPa, and their acoustic responses were tracked by monitoring both second harmonic and subharmonic emissions. Microbubbles were first acoustically characterized with their original gas and then re-characterized after substitution of the original gas with air, SF 6 or C 4 F 10 . A measureable change in intensity of the subharmonic emissions with a 20- to 40-min delayed onset and increasing subharmonic emissions of the order 12-18 dB was recorded for microbubbles filled with C 4 F 10 . Substitution of C 4 F 10 with air eliminated the earlier observed delay in subharmonic emissions. Significantly, substitution of SF 6 for C 4 F 10 successfully triggered a delay in the subharmonic emissions of the resultant agents, whereas substitution of C 4 F 10 for SF 6 eliminated the earlier observed suppression of subharmonic emissions, clearly suggesting that the type of filling gas contained in the microbubble agent influences subharmonic emissions in a time-dependent manner. Because our agents were dispersed in air-stabilized phosphate-buffered saline, these results suggest that the diffusivity of the gas from the agent to the surrounding medium is correlated with the time-dependent evolution of subharmonic emissions., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Phase-shift perfluorocarbon droplets have been investigated for over 20 years as pre-clinical ultrasound contrast agents with distinctive advantages in imaging and therapy. A number of formulation strategies exist, each with inherent advantages and limitations. In this note, we demonstrate a unique opportunity: that phase-shift droplets can be generated directly from commercially available microbubbles. This may facilitate pre-clinical and translational development by reducing the in-house synthesis expertise and resources required to generate high concentration droplet emulsions. Proof-of-principle in vitro and in vivo is given using droplets created from Definity and MicroMarker. The results demonstrate the role of perfluorocarbon choice in the trade-off between thermal stability and vaporization threshold, and suggest that commercial microbubbles with decafluorobutane cores may be ideal for this approach., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Therapeutic microbubbles could make an important contribution to the diagnosis and treatment of cancer. Acoustic characterisation was performed on microfluidic generated microbubble populations that either were bare or had liposomes attached. Through the use of broadband attenuation techniques (3-8 MHz), the shell stiffness was measured to be 0.72 ± 0.01 and 0.78 ± 0.05 N/m and shell friction was 0.37 ± 0.05 and 0.74 ± 0.05 × 10 -6  kg/s for bare and liposome-loaded microbubbles, respectively. Acoustic scatter revealed that liposome-loaded microbubbles had a lower subharmonic threshold, occurring from a peak negative pressure of 50 kPa, compared with 200 kPa for equivalent bare microbubbles. It was found that liposome loading had a negligible effect on the destruction threshold for this microbubble type, because at a mechanical index >0.4 (570 kPa), 80% of both populations were destroyed., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Coronary intervention for myocardial infarction often results in microvascular embolization of thrombus. Sonoreperfusion therapy (SRP) using ultrasound and microbubbles restored perfusion in our in vitro flow model of microvascular obstruction. In this study, we assessed SRP efficacy using whole blood as the perfusate with and without tissue plasminogen activator (tPA). In a phantom vessel bearing a 40-μm-pore mesh to simulate the microvasculature, microthrombi were injected to cause microvascular obstruction and were treated using SRP. Without tPA, the lytic rate increased from 2.6 ± 1.5 mmHg/min with 1000-cycle pulses to 7.3 ± 3.2 mmHg/min with 5000-cycle ultrasound pulses (p < 0.01). The lytic index was similar for tPA-only ([2.0 ± 0.5] × 10 -3  mmHg -1  min -1 ) and 5000 cycles without tPA ([2.3 ± 0.5] × 10 -3  mmHg -1  min -1 ) (p = 0.5) but increased ([3.6 ± 0.8] × 10 -3  mmHg -1  min -1 ) with tPA in conjunction with 5000-cycles ultrasound (p < 0.01). In conclusion, SRP restored microvascular perfusion in whole blood, SRP lytic rate in experiments without tPA increased with ultrasound pulse length and efficacy increased with the addition of tPA., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

High-mechanical-index ultrasound and intravenous microbubbles might prove beneficial in treating microvascular obstruction caused by microthrombi after primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). Experiments in animals have revealed that longer-pulse-duration ultrasound is associated with an improvement in microvascular recovery. This trial tested long-pulse-duration, high-mechanical-index ultrasound in STEMI patients. Non-randomly assigned, non-blinded patients were included in this phase 2 trial. The primary endpoint was any side effect possibly related to the ultrasound treatment. The study was aborted after six patients were included; three patients experienced coronary vasoconstriction of the culprit artery, unresponsive to nitroglycerin. Therefore, coronary artery diameter was measured in five pigs. Coronary artery diameters distal to the injury site decreased after application of ultrasound, after balloon injury plus thrombus injection (from 1.89 ± 0.24 mm before to 1.78 ± 0.17 after ultrasound, p = 0.05). Long-pulse-duration ultrasound might cause coronary vasoconstriction distal to the culprit vessel location., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Phase-shift perfluorocarbon droplets are designed to convert from the liquid to the gas state by the external application of acoustic or optical energy. Although droplet vaporization has been investigated extensively at ultrasonic frequencies between 1 and 10 MHz, few studies have characterized performance at the higher frequencies commonly used in small animal imaging. In this study, we use standard B-mode imaging sequences on a pre-clinical ultrasound platform to both image and activate sub-micron decafluorobutane droplet populations in vitro and in vivo at center frequencies in the range of 20-40 MHz. Results show that droplets remain stable against vaporization at low imaging pressures but are vaporized at peak negative pressures near 3.5 MPa at the three frequencies tested. This study also found that a small number of size outliers present in the distribution can greatly influence droplet performance. Removal of these outliers results in a more accurate assessment of the vaporization threshold and produces free-flowing microbubbles upon vaporization in the mouse kidney., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Ultrasound (US)-microbubble (MB)-mediated therapies have been found to restore perfusion and enhance drug/gene delivery. On the presumption that MBs do not persist during long US exposure under high acoustic pressures, most schemes use short US pulses when a high US pressure is employed. However, we recently observed an enhanced thrombolytic effect using long US pulses at high acoustic pressures. Therefore, we explored the fate of MBs during long tone-burst exposures (5 ms) at various acoustic pressures and MB concentrations via direct high-speed optical observation and passive cavitation detection. MBs first underwent stable or inertial cavitation depending on the acoustic pressure and then formed gas-filled clusters that continued to oscillate, break up and form new clusters. Cavitation detection confirmed continued, albeit diminishing, acoustic activity throughout the 5-ms US excitation. These data suggest that persisting cavitation activity during long tone bursts may confer additional therapeutic effects., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of -28.3, -4.2 and -9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave -8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation., (Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Numerous age-related pathologies affect the prostate gland, the most menacing of which is prostate cancer (PCa). The diagnostic tools for prostate investigation are invasive, requiring biopsies when PCa is suspected. Novel dynamic contrast-enhanced ultrasound (DCE-US) imaging approaches have been proposed recently and appear promising for minimally invasive localization of PCa. Ultrasound imaging of the prostate is traditionally performed with a transrectal probe because the location of the prostate allows for high-resolution images using high-frequency transducers. However, DCE-US imaging requires lower frequencies to induce bubble resonance and, thus, improve contrast-to-tissue ratio. For this reason, in this study we investigate the feasibility of quantitative DCE-US imaging of the prostate via the abdomen. The study included 10 patients (age = 60.7 ± 5.7 y) referred for a needle biopsy study. After having given informed consent, patients underwent DCE-US with both transabdominal and transrectal probes. Time-intensity contrast curves were derived using both approaches and their model-fit quality was compared. Although further improvements are expected by optimization of the transabdominal settings, the results of transabdominal and transrectal DCE-US are closely comparable, confirming the feasibility of transabdominal DCE-US; transabdominal curve fitting revealed an average determination coefficient r(2) = 0.91 (r(2) > 0.75 for 78.6% of all prostate pixels) compared with r(2) = 0.91 (r(2) > 0.75 for 81.6% of all prostate pixels) by the transrectal approach. Replacing the transrectal approach with more acceptable transabdominal scanning for prostate investigation is feasible. This approach would improve patient comfort and represent a useful option for PCa localization and monitoring., (Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The use of ultrasound and microbubbles as an effective adjuvant to thrombolytics has been reported in vitro, ex vivo and in vivo. However, the specific mechanisms underlying ultrasound-enhanced thrombolysis have yet to be elucidated. We present visual observations illustrating two mechanisms of ultrasound-enhanced thrombolysis: acoustic cavitation and radiation force. An in vitro flow model was developed to observe human whole blood clots exposed to human fresh-frozen plasma, recombinant tissue-type plasminogen activator (0, 0.32, 1.58 or 3.15 μg/mL) and the ultrasound contrast agent Definity (2 μL/mL). Intermittent, continuous-wave ultrasound (120 kHz, 0.44 MPa peak-to-peak pressure) was used to insonify the perfusate. Ultraharmonic emissions indicative of stable cavitation were monitored with a passive cavitation detector. The clot was observed with an inverted microscope, and images were recorded with a charge-coupled device camera. The images were post-processed to determine the time-dependent clot diameter and root-mean-square velocity of the clot position. Clot lysis occurred preferentially surrounding large, resonant-sized bubbles undergoing stable oscillations. Ultraharmonic emissions from stable cavitation were found to correlate with the lytic rate. Clots were observed to translate synchronously with the initiation and cessation of the ultrasound exposure. The root-mean-square velocity of the clot correlated with the lytic rate. These data provide visual documentation of stable cavitation activity and radiation force during sub-megahertz sonothrombolysis. The observations of this study suggest that the process of clot lysis is complex, and both stable cavitation and radiation force are mechanistically responsible for this beneficial bio-effect in this in vitro model., (Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

The acoustic characteristics of microbubbles created from vaporized submicron perfluorocarbon droplets with fluorosurfactant coating are examined. Utilizing ultra-high-speed optical imaging, the acoustic response of individual microbubbles to low-intensity diagnostic ultrasound was observed on clinically relevant time scales of hundreds of milliseconds after vaporization. It was found that the vaporized droplets oscillate non-linearly and exhibit a resonant bubble size shift and increased damping relative to uncoated gas bubbles due to the presence of coating material. Unlike the commercially available lipid-coated ultrasound contrast agents, which may exhibit compression-only behavior, vaporized droplets may exhibit expansion-dominated oscillations. It was further observed that the non-linearity of the acoustic response of the bubbles was comparable to that of SonoVue microbubbles. These results suggest that vaporized submicron perfluorocarbon droplets possess the acoustic characteristics necessary for their potential use as ultrasound contrast agents in clinical practice., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Characterizing the non-linear response of microbubble contrast agents is important for their efficacious use in imaging and therapy. In this article, we report that the subharmonic and ultraharmonic response of lipid-shelled microbubble contrast agents exhibits a strong temporal dependence. We characterized non-linear emissions from Targestar-p microbubbles (Targeson Inc., San Diego, CA, USA) periodically for 60 min, at 10 MHz excitation frequency. The results revealed a considerable increase in the subharmonic and ultraharmonic response (nearly 12-15 and 5-8 dB) after 5-10 min of agent preparation. However, the fundamental and the harmonic response remained almost unchanged in this period. During the next 50 min, the subharmonic, fundamental, ultraharmonic, and harmonic responses decreased steadily by 2-5 dB. The temporal changes in the non-linear behavior of the agent appeared to be primarily mediated by gas-exchange through the microbubble shell; temperature and prior acoustic excitation based mechanisms were ruled out. Further, there was no measurable change in the agent size distribution by static diffusion. We envisage that these findings will help obtain reproducible measurements from agent characterization, non-linear imaging, and fluid-pressure sensing. These findings also suggest the possibility for improving non-linear imaging by careful design of ultrasound contrast agents., (Published by Elsevier Inc.)

The aim of this study was to characterize the frequency-dependent acoustic attenuation of three phospholipid-shelled ultrasound contrast agents (UCAs): Definity, MicroMarker and echogenic liposomes. A broadband through-transmission technique allowed for measurement over 2 to 25 MHz with a single pair of transducers. Viscoelastic shell parameters of the UCAs were estimated using a linearized model developed by N. de Jong, L. Hoff, T. Skotland and N. Bom (Ultrasonics 1992; 30:95-103). The effect of diluent on the attenuation of these UCA suspensions was evaluated by performing attenuation measurements in 0.5% (w/v) bovine serum albumin and whole blood. Changes in attenuation and shell parameters of the UCAs were investigated at room temperature (25°C) and physiologic temperature (37°C). The attenuation of the UCAs diluted in 0.5% (w/v) bovine serum albumin was found to be identical to the attenuation of UCAs in whole blood. For each UCA, attenuation was higher at 37°C than at 25°C, underscoring the importance of conducting characterization studies at physiologic temperature. Echogenic liposomes exhibited a larger increase in attenuation at 37°C versus 25°C than either Definity or MicroMarker., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

In contrast to the clinically used microbubble ultrasound contrast agents, nanoscale bubbles (or nanobubbles) may potentially extravasate into tumors that exhibit more permeable vasculature, facilitating targeted molecular imaging and drug delivery. Our group recently presented a simple strategy using the non-ionic surfactant Pluronic as a size control excipient to produce nanobubbles with a mean diameter of 200 nm that exhibited stability and echogenicity on par with microbubbles. The objective of this study was to carry out an in-depth characterization of nanobubble properties as compared with Definity microbubbles, both in vitro and in vivo. Through use of a tissue-mimicking phantom, in vitro experiments measured the echogenicity of the contrast agent solutions and the contrast agent dissolution rate over time. Nanobubbles were found to be more echogenic than Definity microbubbles at three different harmonic frequencies (8, 6.2 and 3.5 MHz). Definity microbubbles also dissolved 1.67 times faster than nanobubbles. Pharmacokinetic studies were then performed in vivo in a subcutaneous human colorectal adenocarcinoma (LS174T) in mice. The peak enhancement and decay rates of contrast agents after bolus injection in the liver, kidney and tumor were analyzed. No significant differences were observed in peak enhancement between the nanobubble and Definity groups in the three tested regions (tumor, liver and kidney). However, the decay rates of nanobubbles in tumor and kidney were significantly slower than those of Definity in the first 200-s fast initial phase. There were no significant differences in the decay rates in the liver in the initial phase or in three regions of interest in the terminal phase. Our results suggest that the stability and acoustic properties of the new nanobubble contrast agents are superior to those of the clinically used Definity microbubbles. The slower washout of nanobubbles in tumors suggests potential entrapment of the bubbles within the tumor parenchyma., (Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Angiogenesis plays a major role in prostate cancer growth. Despite extensive research on blood perfusion imaging aimed at angiogenesis detection, the diagnosis of prostate cancer still requires systematic biopsies. This may be due to the complex relationship between angiogenesis and microvascular perfusion. Analysis of ultrasound-contrast-agent dispersion kinetics, determined by multipath trajectories in the microcirculation, may provide better characterization of the microvascular architecture. We propose the physical rationale for dispersion estimation by an existing spatiotemporal similarity analysis. After an intravenous ultrasound-contrast-agent bolus injection, dispersion is estimated by coherence analysis among time-intensity curves measured at neighbor pixels. The accuracy of the method is increased by time-domain windowing and anisotropic spatial filtering for speckle regularization. The results in 12 patient data sets indicated superior agreement with histology (receiver operating characteristic curve area = 0.88) compared with those obtained by reported perfusion and dispersion analyses, providing a valuable contribution to prostate cancer localization., (Copyright © 2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)