An in vitro study of a microbubble contrast agent using a clinical ultrasound imaging system

Optimal insonation settings for contrast imaging are yet to be specified, mainly due to the lack of good understanding of the behaviour of the microbubbles. A satisfactory model that explains the behaviour of individual contrast agent scatterers has not yet been reported in the literature. An in vitro system based on a commercial scanner (ATL HDI3000) has been developed to investigate the backscatter of such agents. Suspensions of Definity were introduced in an anechoic tank. The frequency of transmitted ultrasound varied from 1 to 5 MHz, pulse period from 2 to 10 periods and peak negative acoustic pressure from 0.08 to 1.7 MPa. The backscatter at the fundamental and second harmonic frequency windows from the agent was normalized in terms of the corresponding components of backscatter from a blood mimicking fluid suspension. The agent provided a dominant resonance effect at 1.6 MHz transmit frequency. Second harmonic normalized backscatter averaged around 9 dB higher than the fundamental. The normalized fundamental backscatter intensity was linear with peak negative pressure. The second harmonic at resonance peaked at 0.5 MPa suggestive of bubble disruption above such pressure. The system proved capable of illustrating the ultrasonic behaviour of Definity in vitro, and the investigation suggested particular insonation conditions for optimal image enhancement using Definity.     

In vitro characterization of the subharmonic ultrasound signal from Definity microbubbles at high frequencies

Ultrasound microbubble contrast agents have been demonstrated to scatter subharmonic energy at one-half the driving frequency. At ultrasound frequencies in the 20-40 MHz range, the subharmonic offers the potential to differentiate the blood in the microcirculation from the surrounding tissue. It is unknown whether current contrast agents, manufactured to be resonant between 2 and 12 MHz, are ideal for subharmonic imaging at higher frequencies. We performed numerical simulations of the Keller-Miksis model for the behavior of a single bubble and experimental investigations of Definity microbubbles in water. The results supported the hypothesis that off-resonant bubbles, excited at their second harmonic, may be primarily responsible for the observed subharmonic energy. For frequencies between 20 and 32 MHz and 32 and 40 MHz, the optimal bubble diameters for the generation of subharmonics in vitro were determined experimentally to be 1.2-5 microm and less than 1.2 microm, respectively. Definity may be a suitable ultrasound contrast agent for subharmonic imaging at 20 MHz with peak-negative pressures between 380 and 590 kPa and pulses greater than or equal to four cycles in duration.     

An in vitro system for the study of ultrasound contrast agents using a commercial imaging system.

An in vitro system for the investigation of the behaviour of contrast microbubbles in an ultrasound field, that provides a full diagnostic range of settings, is yet to be presented in the literature. The evaluation of a good compromise of such a system is presented in this paper. It is based on (a) an HD13000 ATL scanner (Bothell, WA, USA) externally controlled by a PC and (b) on the use of well-defined reference materials. The suspensions of the reference ultrasonic scattering material are placed in an anechoic tank. The pulse length ranges from 2 to 10 cycles, the acoustic pressure from 0.08 to 1.8 MPa, the transmit frequency from 1 to 4.3 MHz, and the receive frequency from 1 to 8 MHz. The collection of 256 samples of RF data, at an offset distance from the transducer face, was performed at 20 MHz digitization rate, which corresponds to approximately 1 cm depth in water. Two particle suspensions are also presented for use as reference scatterers for contrast studies: (a) a suspension of Orgasol (ELF Atochem, Paris, France) particles (approximately 5 microm mean diameter) and (b) a suspension of Eccosphere (New Metals & Chemicals Ltd, Essex, UK) particles (approximately 50 microm mean diameter). A preliminary experiment with the contrast agent Definity (DuPont Pharmaceutical Co, Waltham, MA) showed that the above two materials are suitable for use as a reference for contrast backscatter.     

Enhancement of subharmonic emission from encapsulated microbubbles by using a chirp excitation technique

Subharmonic contrast imaging promises to improve ultrasound-imaging quality by taking advantage of an increased contrast to tissue signal. However, acoustic pressures beyond the subharmonic generation threshold using common ultrasound pulses may induce significant contrast microbubble destruction. In this work, a chirp excitation technique is presented to enhance the subharmonic emission from encapsulated microbubbles. Chirp signals with a center frequency of 5 MHz, variable frequency range and duration time are employed to drive microbubbles in numerical simulation and experimental studies. We provide a theoretical evaluation of the chirp excitation pressure threshold and the acoustic pressure dependence of subharmonic based on Church's model and demonstrate that the amplitude and axial resolution of the subharmonic can be optimized by proper selection of the frequency range and time duration of the chirp signal. Measurements are qualitatively in agreement with the simulation. Moreover, we demonstrate that chirp excitation may be able to improve the amplitude of the subharmonic component up to 22 dB over the pulse excitation. The chirp excitation technique could potentially be used for improving the subharmonic contrast imaging quality.     

Characterization of individual ultrasound microbubble dynamics with a light-scattering system

Ultrasound microbubbles are contrast agents used for diagnostic ultrasound imaging and as carriers for noninvasive payload delivery. Understanding the acoustic properties of individual microbubble formulations is important for optimizing the ultrasound imaging parameters for improved image contrast and efficient payload delivery. We report here a practical and simple optical tool for direct real-time characterization of ultrasound contrast microbubble dynamics based on light scattering. Fourier transforms of raw linear and nonlinear acoustic oscillations, and microbubble cavitations are directly recorded. Further, the power of this tool is demonstrated by comparing clinically relevant microbubble cycle-to-cycle dynamics and their corresponding Fourier transforms.     

Subharmonic generation from ultrasonic contrast agents

Ultrasonic contrast agents are used to enhance backscatter from blood and thus aid in delineating blood from surrounding tissue. However, behaviour of contrast agents in an acoustic field is nonlinear and leads to harmonic components in the backscattered signal. Various research groups have investigated second-harmonic emissions. In this work, the subharmonic emission from contrast agents is investigated with a view towards potential use in imaging. It is shown that the microbubbles with various surface properties, such as contrast agents, generate significant subharmonics under various insonating conditions. Theoretical results as well as experimental results using Optison indicate the generation of strong subharmonics with burst insonation at twice the resonant frequency of the microbubble. It is suggested that subharmonic imaging may provide a better modality than second-harmonic imaging to delineate blood from tissue and will be of significant importance for imaging deep vessels, such as in echocardiography and vascular diseases, due to the high signal-to-clutter ratio of the subharmonic imaging.     

Superparamagnetic iron oxide nanoparticle-embedded encapsulated microbubbles as dual contrast agents of magnetic resonance and ultrasound imaging

An encapsulated microbubble (EMB) of a novel construct is proposed to enhance the magnetic resonance imaging contrast by introducing superparamagnetic iron oxide (SPIO) nanoparticles (mean diameter is 12 nm) into the polymer shell of the microbubble. Such microbubble vesicle has nitrogen gas in the core and its mean diameter is 3.98 μm. An in vitro MR susceptibility experiment using a phantom consisting EMBs has shown that the relationship between the transverse relaxation rate R₂ and the Fe₃O₄ nanoparticle concentration in the shell (the volume fraction of EMBs is kept constant) can be fitted to a linear function and an exponentially growth function is observed between R₂ and the SPIO-inclusion microbubble concentration. The in vivo MRI experiments also show that the SPIO-inclusion microbubbles have longer contrast-enhancement duration time in rat liver than non-SPIO-inclusion microbubbles. An in vitro ultrasound imaging experiment of SPIO-inclusion microbubbles also shows that they can enhance the ultrasound contrast significantly. Additionally, the interaction between the SPIO-inclusion microbubbles and cells indicates that such microbubble construct can retain the acoustic property under the ultrasound exposure by controlling the SPIO concentration in the shell. Therefore, the proposed SPIO nanoparticle-embedded EMBs potentially can become effective MR susceptibility contrast agents while also can be good US contrast agents.     

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents.     

基质细胞衍生因子1靶向超声对比剂在体内可与血管内皮细胞紧密结合

背景：基质细胞衍生因子1是心肌梗死区域微环境中效力最强的趋化因子,在趋化干细胞修复梗死心肌以及在促进血管新生方面起到重要的作用。微泡和声学活性物质携带靶向配基,可制备成超声成像靶向对比剂并与活体细胞结合,用于分子成像,超声分子成像的关键是寻找“成像靶点”,并成功制备能与“成像靶点” 特异、高效结合的靶向超声对比剂。 目的：实验制备和评价携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂。 方法：采用“生物素-亲和素”桥接法构建携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂,并从外观、pH值、粒径测定、光镜及荧光显微镜下观、流式细胞仪检测等多个方面对靶向对比剂进行评价。4头中华小型猪均结扎左冠状动脉前降支第一对角支制备心肌梗死模型,2头开胸但不结扎左冠状动脉前降支第一对角支,均注入靶向超声对比剂,心肌组织冰冻切片后采用免疫荧光法检测靶向微泡的体内稳定性。 结果与结论：通过生物素-亲和素桥接法可将基质细胞衍生因子1抗体和超声微泡两者结合。体外实验中对比剂外观：表现为半透明的淡黄或绿色,静置后分层。非靶向对比剂pH值为7.02±0.12,靶向微泡对比剂的pH值为6.10±0.19。荧光显微镜下观察靶向微泡明亮且呈指环状绿色荧光环绕外壳周边,剧烈震荡后表面荧光无明显改变。靶向对比剂在携带基质细胞衍生因子1抗体之后微泡粒径大小为(2 422.62±238.82) nm。流式细胞仪检测显示,靶向对比剂在不同时间段的基质细胞衍生因子1携带率稳定,静置1 h后携带率稳定且剧烈震荡前后差异无显著性意义。在体内实验中可见靶向微泡在心梗部位血管内皮细胞处聚集。结果证实,经生物素-亲和素桥接法制备的携基质细胞衍生因子1单克隆抗体靶向微泡超声对比剂体内可与血管内皮细胞结合,在体外结合率高而且结合稳定。     

超声对比剂的材料学特点及临床应用

背景：目前临床上应用的超声对比剂均是含有不同包膜材料和气体成分的微泡对比剂,微泡对比剂的出现使超声诊断技术得到了较大的发展。 目的：探讨超声对比剂的材料学研究特点,及超声对比剂在临床疾病治疗中的应用。 方法：超声对比剂是由气体微泡和外部包裹的膜物质组成,包膜材料主要分为白蛋白、大分子脂质体、多聚体和各种表面活性剂等。超声造影是通过增强背向散射信号来成像。超声对比剂研究的发展大致分为3个阶段,造影相关技术包括二次谐波、组织特异性显像、反相脉冲谐波成像、相干造影成像技术、对比脉冲序列、能量多普勒谐波成像、间歇谐波成像技术、编码谐波成像和超声造影三维成像。 结果与结论：超声对比剂形式的不同主要是通过改变微泡包膜和气体的性质和设计来实现的。微气泡能够实现超声对比剂的显影作用,还可在药物传输上发挥功能。新型的微泡超声对比剂不仅可以提供血流灌注学信息,还可以通过靶向作用于病变组织,分析病变的发生机制,使微泡对比剂的诊断更准确。随着微泡对比剂材料学研究以及制备工艺完善,使超声对比剂具有良好的生物相容性。不仅可以用于各种状态下的特异性超声造影,还可以利用空化效应携带药物或治疗基因向目标组织转移释放。微泡造影技术具有治疗、诊断和超声成像的功能,是一种安全、高效、无创的诊断和靶向传输治疗手段。     

Preparation of targeted microbubbles: ultrasound contrast agents for molecular imaging

Targeted ultrasound contrast agents can be prepared by attaching targeting ligands to the lipid, protein or polymer shell coating of gas-filled microbubbles. These materials are stable on storage, fully biocompatible and can be administered parenterally. Detection of microbubble contrast agents by ultrasound is very efficient (single particles with picogram mass can be visualized). Covalent or noncovalent binding techniques can be used to attach targeting ligands. Ligand-carrying microbubbles adhere to the respective molecular targets in vitro and in vivo. Several biomechanical methods are available to improve targeting efficacy, such as the use of a flexible tether spacer arm between the ligand and the bubble, and the use of folds on the microbubble shell, that project out, enhancing the contact area and increasing the length of the lever arm.     

Threshold of fragmentation for ultrasonic contrast agents

Ultrasound contrast agents are small microbubbles that can be readily destroyed with sufficient acoustic pressure, typically, at a frequency in the low megaHertz range. Microvascular flow rate may be estimated by destroying the contrast agent in a vascular bed, and estimating the rate of flow of contrast agents back into the vascular bed. Characterization of contrast agent destruction provides important information for the design of this technique. In this paper, high-speed optical observation of an ultrasound contrast agent during acoustic insonation is performed. The resting diameter is shown to be a significant parameter in the prediction of microbubble destruction, with smaller diameters typically correlated with destruction. Pressure, center frequency, and transmission phase are each shown to have a significant effect on the fragmentation threshold. A linear prediction for the fragmentation threshold as a function of pressure, when normalized by the resting diameter, has a rate of change of 300 kPa/microm for the range of pressures from 310 to 1200 kPa, and a two-cycle excitation pulse with a center frequency of 2.25 MHz. A linear prediction for the fragmentation threshold as a function of frequency, when normalized by the resting diameter, has a rate of change of -1.2 MHz/microm for a transmission pressure of 800 kPa, and a two-cycle excitation pulse with a range of frequencies from 1 to 5 MHz.     

A novel microbubble construct for intracardiac or intravascular MR manometry: a theoretical study

It has been demonstrated that gas-filled microbubble contrast agents, based on their volume changes, can serve as pressure probes in an MR field. It was recently reported that such an MR-based pressure measurement with microbubbles at 1.5 T must make use of microbubbles that have a volumetric magnetic susceptibility difference with the blood of at least 34 ppm in SI units. In this work, we show through analytical approximations and numerical simulations that such a microbubble formulation can be achieved by coating typical lipid-shelled microbubbles with particles of high dipole moment. Through finite-element simulations we demonstrate that the effective volumetric magnetic susceptibility of a coated microbubble is dependent on the radius, the shell volume fraction and the magnetic susceptibility of the particulates on the shell. Our calculations suggest that a suitable microbubble formulation which will be MR-sensitive to small pressure changes at 1.5 T must be 2-3 microm in radius and be uniformly coated with single-domain magnetic nanoparticles, such as magnetite, at shell volume fractions below 5%.     

Unbinding of targeted ultrasound contrast agent microbubbles by secondary acoustic forces

Targeted molecular imaging with ultrasound contrast agent microbubbles is achieved by incorporating targeting ligands on the bubble coating and allows for specific imaging of tissues affected by diseases. Improved understanding of the interplay between the acoustic forces acting on the bubbles during insonation with ultrasound and other forces (e.g. shear due to blood flow, binding of targeting ligands to receptors on cell membranes) can help improve the efficacy of this technique. This work focuses on the effects of the secondary acoustic radiation force, which causes bubbles to attract each other and may affect the adhesion of targeted bubbles. First, we examine the translational dynamics of ultrasound contrast agent microbubbles in contact with (but not adherent to) a semi-rigid membrane due to the secondary acoustic radiation force. An equation of motion that effectively accounts for the proximity of the membrane is developed, and the predictions of the model are compared with experimental data extracted from optical recordings at 15 million frames per second. A time-averaged model is also proposed and validated. In the second part of the paper, initial results on the translation due to the secondary acoustic radiation force of targeted, adherent bubbles are presented. Adherent bubbles are also found to move due to secondary acoustic radiation force, and a restoring force is observed that brings them back to their initial positions. For increasing magnitude of the secondary acoustic radiation force, a threshold is reached above which the adhesion of targeted microbubbles is disrupted. This points to the fact that secondary acoustic radiation forces can cause adherent bubbles to detach and alter the spatial distribution of targeted contrast agents bound to tissues during activation with ultrasound. While the details of the rupture of intermolecular bonds remain elusive, this work motivates the use of the secondary acoustic radiation force to measure the strength of adhesion of targeted microbubbles.     

Theoretical predictions of harmonic generation from submicron ultrasound contrast agents for nonlinear biomedical ultrasound imaging

Submicron ultrasound contrast agents have aroused attention for their significant promise in ultrasonic contrast/molecular imaging, targeted therapy and echo particle imaging velocimetry. However, nonlinear acoustic properties of submicron encapsulated gas bubbles for ultrasonic applications are still not clearly understood. In this paper, nonlinear acoustic emission characteristics from submicron bubbles were examined using a numerical study. The modified RP equation incorporating viscosity, acoustic radiation, thermal effects and encapsulated shell was used to study single bubble dynamics. Further, a size integration method, shown previously to be useful in prediction of backscatter spectra from groups of bubbles, was applied to analyse response from a bubble population. We show that bubbles with radii (200-500 nm) produce significant subharmonic and ultraharmonic components of the backscatter spectrum, while smaller bubbles (<200 nm) provide substantial second harmonic components. Additionally, nanoscale bubbles (<100 nm) produce very low backscatter amplitudes and thus may not be useful with the use of current ultrasound technology. Analysing optimal ultrasound driving pressures and bubbles size ranges for maximal subharmonic and ultraharmonic signals showed that sub and ultraharmonic mode nonlinear imaging methods may be potentially competitive for larger size bubbles (>200 nm) in providing proper contrast-to-tissue signal ratios.     

Novel ultrasound contrast agent based on microbubbles generated from surfactant mixtures of Span 60 and polyoxyethylene 40 stearate

In this study, novel perfluorocarbon-filled microbubbles as ultrasound contrast agent were fabricated using ultrasonication of a surfactant mixture of sorbitan monostearate (Span 60) and polyoxyethylene 40 stearate (PEG40S) in aqueous media. The microbubbles generated from a 1:9 mixture of PEG40S/Span 60 exhibited an average diameter of 2.08 ± 1.27 μm. More than 99% of the microbubbles had a mean particle diameter less than 8 μm, indicating that they were appropriately sized for intravenous administration as ultrasound contrast agent. The stabilization mechanism of the microbubbles was investigated by the Langmuir–Blodgett technique including the measurements of surface pressure–area (π–A) isotherms and compression–decompression cycles with a two-dimensional monolayer of Span 60 and PEG40S. The dependence on molar fraction of PEG40S in π–A isotherms of mixed monolayers provided a strong evidence of interactions between the two microbubble-forming materials. It is suggested that the monolayer shell imparts good stability to the microbubbles by three means: (1) a low surface tension monolayer hinders dissolution through the reduction of surface tension, which introduces a mechanical surface pressure that counters the Laplace pressure; (2) the presence of a monolayer shell imparts a significant barrier to gas escaping from the core into the aqueous medium; and (3) encapsulation elasticity stabilizes microbubbles against diffusion-driven dissolution and explains the long shelf-life of microbubble contrast agent. The preliminary in vivo ultrasound imaging study showed that such stabilized microbubbles demonstrated excellent enhancement under grey-scale pulse inversion harmonic imaging and power Doppler imaging.     

Selective imaging of adherent targeted ultrasound contrast agents

The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an 'image-push-image' pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging.     

Acoustic response of compliable microvessels containing ultrasound contrast agents

The existing models of the dynamics of ultrasound contrast agents (UCAs) have largely been focused on an UCA surrounded by an infinite liquid. Preliminary investigations of a microbubble's oscillation in a rigid tube have been performed using linear perturbation, under the assumption that the tube diameter is significantly larger than the UCA diameter. In the potential application of drug and gene delivery, it may be desirable to fragment the agent shell within small blood vessels and in some cases to rupture the vessel wall, releasing drugs and genes at the site. The effect of a compliant small blood vessel on the UCA's oscillation and the microvessel's acoustic response are unknown. The aim of this work is to propose a lumped-parameter model to study the interaction of a microbubble oscillation and compliable microvessels. Numerical results demonstrate that in the presence of UCAs, the transmural pressure through the blood vessel substantially increases and thus the vascular permeability is predicted to be enhanced. For a microbubble within an 8 to 40 microm vessel with a peak negative pressure of 0.1 MPa and a centre frequency of 1 MHz, small changes in the microbubble oscillation frequency and maximum diameter are observed. When the ultrasound pressure increases, strong nonlinear oscillation occurs, with an increased circumferential stress on the vessel. For a compliable vessel with a diameter equal to or greater than 8 microm, 0.2 MPa PNP at 1 MHz is predicted to be sufficient for microbubble fragmentation regardless of the vessel diameter; however, for a rigid vessel 0.5 MPa PNP at 1 MHz may not be sufficient to fragment the bubbles. For a centre frequency of 1 MHz, a peak negative pressure of 0.5 MPa is predicted to be sufficient to exceed the stress threshold for vascular rupture in a small (diameter less than 15 microm) compliant vessel. As the vessel or surrounding tissue becomes more rigid, the UCA oscillation and vessel dilation decrease; however the circumferential stress is predicted to increase. Decreasing the vessel size or the centre frequency increases the circumferential stress. For the two frequencies considered in this work, the circumferential stress does not scale as the inverse of the square root of the acoustic frequency va as in the mechanical index, but rather has a stronger frequency dependence, 1/va.     

白蛋白微泡促进报告基因在细胞表达的实验研究

目的: 探讨白蛋白微泡作为非病毒载体在基因传输中的作用。方法:6孔板培养脐静脉内皮细胞(EC)和血管平滑肌细胞(VSMC),每孔加pcDNA3.1/His/LacZ质粒20 μg,加不同浓度微泡或不加微泡,超声条件为连续波,频率2MHz,机械指数1.8,照射时间1 min,48 h后计算蓝染细胞百分率和β-半乳糖苷酶活性。另以不同浓度微泡及超声照射时间处理细胞,测定细胞增殖情况。结果:与单纯超声质粒组相比,含微泡组蓝染细胞率增加约10-15倍(其中内皮细胞组11.6倍,平滑肌细胞组15.2倍),报告基因表达定量增加近8倍。微泡浓度为10％时细胞转染效率最高。超声照射对细胞增殖无影响,微泡浓度为50％时有明显的细胞毒作用。结论:白蛋白微泡在超声作用下能明显增加基因的传输效率,有可能成为一种安全有效的基因治疗的载体。     

Safety and bio-effects of ultrasound contrast agents

The use of gas-filled microbubbles as ultrasound contrast agents raises potential safety concerns for diagnostic ultrasound imaging. A number of biological effects have been seen in experimental systems, including the induction of physiological response to cardiac exposures (premature ventricular contractions) and damage at a microvascular level (microvascular rupture and petechial haemorrhage). The literature indicates that a mechanical index (MI) of 0.4 represents the threshold above which microvascular bio-effects are seen in in vivo studies. Above this value, the extent of biological effects appears to increase rapidly with both increasing in situ peak negative acoustic pressure amplitude and with contrast agent concentration. While there is no proven evidence of harm resulting from clinical use of these agents, caution is recommended when contrast-enhanced imaging is undertaken.