Combining Subharmonic and Ultraharmonic Modes for Intravascular Ultrasound Imaging: A Preliminary Evaluation

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.     

Impact of Filling Gas on Subharmonic Emissions of Phospholipid Ultrasound Contrast Agents

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 (SF₆), octafluoropropane (C₃F₈) or decafluorobutane (C₄F₁₀), nitrogen (N₂)/C₄F₁₀ 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, SF₆ or C₄F₁₀. 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 C₄F₁₀. Substitution of C₄F₁₀ with air eliminated the earlier observed delay in subharmonic emissions. Significantly, substitution of SF₆ for C₄F₁₀ successfully triggered a delay in the subharmonic emissions of the resultant agents, whereas substitution of C₄F₁₀ for SF₆ 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.     

Subharmonic,Non-linear Fundamental and Ultraharmonic Imaging of Microbubble Contrast at High Frequencies

There is increasing use of ultrasound contrast agent in high-frequency ultrasound imaging. However, conventional contrast detection methods perform poorly at high frequencies. We performed systematic in vitro comparisons of subharmonic, non-linear fundamental and ultraharmonic imaging for different depths and ultrasound contrast agent concentrations (Vevo 2100 system with MS250 probe and MicroMarker ultrasound contrast agent, VisualSonics, Toronto, ON, Canada). We investigated 4-, 6- and 10-cycle bursts at three power levels with the following pulse sequences: B-mode, amplitude modulation, pulse inversion and combined pulse inversion/amplitude modulation. The contrast-to-tissue (CTR) and contrast-to-artifact (CAR) ratios were calculated. At a depth of 8 mm, subharmonic pulse-inversion imaging performed the best (CTR = 26 dB, CAR = 18 dB) and at 16 mm, non-linear amplitude modulation imaging was the best contrast imaging method (CTR = 10 dB). Ultraharmonic imaging did not result in acceptable CTRs and CARs. The best candidates from the in vitro study were tested in vivo in chicken embryo and mouse models, and the results were in a good agreement with the in vitro findings.     

Characterisation of Liposome-Loaded Microbubble Populations for Subharmonic Imaging

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.     

A Review of Phospholipid Encapsulated Ultrasound Contrast Agent Microbubble Physics

Ultrasound contrast agent microbubbles have expanded the utility of biomedical ultrasound from anatomic imaging to the assessment of microvascular blood flow characteristics and ultrasound-assisted therapeutic applications. Central to their effectiveness in these applications is their resonant and non-linear oscillation behaviour. This article reviews the salient physics of an oscillating microbubble in an ultrasound field, with particular emphasis on phospholipid-coated agents. Both the theoretical underpinnings of bubble vibration and the experimental evidence of non-linear encapsulated bubble dynamics and scattering are discussed and placed within the context of current and emerging applications.     

The Effect of Binding on the Subharmonic Emissions from Individual Lipid-Encapsulated Microbubbles at Transmit Frequencies of 11 and 25 MHz

Targeted microbubble imaging at ultrasound frequencies above 5 MHz has applications in both a preclinical context for a range of disease processes and clinically for the assessment of atherosclerosis and superficial tumors. Although the feasibility of ultrasound molecular imaging has been well demonstrated for a range of target molecules, little work has examined the effects of binding on microbubble oscillations, which is of potential relevance to improving the sensitivity, specificity, and quantification of bound-bubble detection. In this study we investigated the influence of binding on the subharmonic response of bubbles at transmit frequencies of 11 and 25 MHz. Individual bubbles were situated adjacent to a boundary in either a bound or an unbound state, optically sized and acoustically interrogated with pressures ranging from 0.02 to 1.2 MPa. At 11 MHz, unbound bubbles (n = 53) were found to have strong subharmonic activity for sizes between 2.4 and 2.6 μm, whereas bound bubbles (n = 50) were most active from 2.6 to 3.0 μm. Destruction thresholds were found to be lower for bound bubbles. At 25 MHz, bound-bubble (n = 57) activity was found to peak at 1.9 μm as compared to 2.1 μm in the unbound cases (n = 53), with a 20% increase in amplitude. Comparison with simulations indicates that both unbound and bound bubbles undergo compression-only behavior at 11 MHz, and expansion-dominated behavior at 25 MHz. Subharmonic emissions elicited from 0 radian transmit pulses were found to be π/2 radians out of phase with those elicited from a π radian transmit pulse, suggesting inefficient subharmonic preservation from pulse inversion schemes. With the appropriate postprocessed phase correction, an increase in the subharmonic amplitude of up to 60% was shown, depending on the bubble size and transmit frequency.     

Ultrasound with Microbubble Contrast Agent and Urokinase for Thrombosis

This study was aimed at assessing the effects of urokinase (UK) in combination with ultrasound and microbubbles in in vitro and in vivo thrombolytic therapy for the treatment of deep vein thrombosis (DVT). Thrombi with formation times of 1, 3, 7, 14 and 21 d were used for thrombolysis. Forty-five adult mongrel dogs were used to evaluate thrombosis in vivo. Both in vitro and in vivo analyses revealed that UK?+?microbubbles had the best effect among the combinations. Thrombolysis <7 d was more effective at a thrombolysis rate of about 50%, but the thrombolytic effect of thrombi >7 d was poor at thrombolysis rates <30%. Ultrasound?+?UK significantly increased the thrombolysis rate of thrombi <7 d. These results suggest that the combination of ultrasound with microbubble contrast agents and UK may have a synergistic effect on thrombolysis.     

Microbubble Void Imaging: A Non-invasive Technique for Flow Visualisation and Quantification of Mixing in Large Vessels Using Plane Wave Ultrasound and Controlled Microbubble Contrast Agent Destruction

There is increasing recognition of the influence of the flow field on the physiology of blood vessels and their development of pathology. Preliminary work is reported on a novel non-invasive technique, microbubble void imaging, which is based on ultrasound and controlled destruction of microbubble contrast agents, permitting flow visualisation and quantification of flow-induced mixing in large vessels. The generation of microbubble voids can be controlled both spatially and temporally using ultrasound parameters within the safety limits. Three different model vessel geometries—straight, planar-curved and helical—with known effects on the flow field and mixing were chosen to evaluate the technique. A high-frame-rate ultrasound system with plane wave transmission was used to acquire the contrast-enhanced ultrasound images, and an entropy measure was calculated to quantify mixing. The experimental results were cross-compared between the different geometries and with computational fluid dynamics. The results indicated that the technique is able to quantify the degree of mixing within the different configurations, with a helical geometry generating the greatest mixing, and a straight geometry, the lowest. There is a high level of concordance between the computational fluid dynamics and experimental results. The technique could also serve as a flow visualisation tool.     

Broadband Attenuation Measurements of Phospholipid-Shelled Ultrasound Contrast Agents

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.     

超声空化微气泡造影剂对人乳腺癌细胞影响的实验研究

目的观察不同浓度脂质体微气泡造影剂在超声照射下对体外培养的人乳腺癌细胞的影响。 方法12孔板培养人乳腺癌细胞并分为：（1）对照组；（2）单纯超声照射组；（3）单纯造影剂组；（4）造影剂＋超声照射组。不同造影剂浓度下，声波发射强度一3dB，照射时间60s。给予实验刺激后，原子力显微镜动态观察细胞膜形态的改变，PI染色后观察细胞的活性。 结果单纯超声照射组及造影剂组细胞膜形态与对照组比较无明显改变。造影剂＋超声照射组细胞膜出现大小不等的微孔，提高造影剂浓度，孔径增大，但细胞活性显著下降。 结论微气泡造影剂显著增强超声的空化效应，提高细胞的通透性，对细胞形态及细胞活性的影响与造影剂浓度有关。     

Growth and Dissolution of an Encapsulated Contrast Microbubble: Effects of Encapsulation Permeability

Gas diffusion from an encapsulated microbubble is modeled using an explicit linear relation for gas permeation through the encapsulation. Both the cases of single gas (air) and multiple gases (perfluorocarbon inside the bubble and air dissolved in surrounding liquid) are considered. An analytical expression for the dissolution time for an encapsulated air bubble is obtained; it showed that for small permeability the dissolution time increases linearly with decreasing permeability. A perfluorocarbon-filled contrast microbubble such as Definity® was predicted to experience a transient growth because of air infusion before it dissolves in conformity with previous experimental findings. The growth phase occurs only for bubbles with a critical value of initial mole fraction of perfluorocarbon relative to air. With empirically obtained property values, the dissolution time of a 2.5-micron diameter (same as that of Definity), lipid-coated octafluoropropane bubble, with surface tension 25 mN/m, is predicted to be 42 min in an air-saturated medium. The properties such as shell permeability, surface tension and relative mole fraction of octafluoropropane are varied to investigate their effects on the time scales of bubble growth and dissolution, including their asymptotic scalings where appropriate. The dissolution dynamics scales with permeability, in that when the time is nondimensioanlized with permeability, curves for different permeabilities collapse on a single curve. Investigation of bubbles filled with other gases (nonoctafluoropropane perfluorocarbon and sulfur hexafluoride) indicates longer dissolution time because of lower solubility and lower diffusivity for larger gas molecules. For such micron-size encapsulated bubbles, lifetime of hours is possible only at extremely low surface tension (<1 mN/m) or at extreme oversaturation. (E-mail: sarkar@udel.edu)     

Transcranial contrast diminution imaging of the human brain: a pilot study in healthy volunteers

Analysis of contrast diminution kinetics after bubble destruction is a new aspect in harmonic imaging. The purpose of this study was to investigate this approach to human cerebral perfusion. A total of 12 healthy volunteers were investigated transtemporally (Philips SONOS 5500, S4-probe, 1.8 to 3.6 MHz, 10 cm) at two ultrasound (US) contrast agent (UCA) infusion rates (0.5 and 1.0 mL/min of Optison). After achieving a steady-state, a set of 12 US pulses (6.67 Hz, MI 1.6) was applied. Time-intensity plots of three regions-of-interest (ROIs) (thalamus, white matter and cortex) were analyzed, using an exponential curve fit (I((t)) = I(0)e(-betat) + B). A total of 20 of 20 successful investigations showed a signal decrease after pulsed US application. In all cases, it was possible to generate exponential time-intensity curves. Half-life (T(1/2) = ln2/beta) and baseline intensity (B) showed a significant dependence on infusion rate (p = 0.01). At 1.0 mL/min, T(1/2) also depended on investigation depth (p = 0.01). It is possible to assess contrast diminution kinetics in human cerebral microcirculation. This new approach may provide additional information on cerebral perfusion within a short investigation time.     

Investigating the subharmonic response of individual phospholipid encapsulated microbubbles at high frequencies: a comparative study of five agents

There are a range of contrast ultrasound applications above 10 MHz, a frequency regime in which nonlinear microbubble behavior is poorly understood. Lipid-encapsulated microbubbles have considerable potential for use at higher frequencies because they have been shown to exhibit pronounced nonlinear activity at frequencies up to 40 MHz. The objective of this work was to investigate the influence of agent formulation on the subharmonic response of lipid-encapsulated microbubbles at high frequencies with a view to providing information relevant to improving contrast agent design and imaging performance. An optical-acoustical setup was used to measure the subharmonic emissions from small (d < 3 μm) individual lipid-encapsulated microbubbles as a function of transmit pressure, size and composition. In this study, five agent formulations (Definity™, MicroMarker™ and three in-house agents manipulated to exhibit different levels of shell microstructure heterogeneity) were insonified at 25 MHz over a peak negative pressure (P_n) range of 0.02–1.2 MPa. All agents exhibited distinctly different subharmonic behavior, both in terms of amplitude and active sizes. MicroMarker™ exhibited the strongest, broadest and most consistent subharmonic response, 22% greater in power than that of Definity™ and as much as 50% greater than the in-house formulations. No clear relation between in-house agents’ shell microstructure and nonlinear response was found, other than the variability in the nonlinear response itself. An analysis of the response of MicroMarker™ bubbles suggests that these bubbles exhibit “expansion-dominated” oscillations, in contrast to “compression-only” oscillations observed for similar bubbles at lower frequencies (f < 11 MHz).     

Flow Velocity Mapping Using Contrast Enhanced High-Frame-Rate Plane Wave Ultrasound and Image Tracking: Methods and Initial in Vitro and in Vivo Evaluation

Ultrasound imaging is the most widely used method for visualising and quantifying blood flow in medical practice, but existing techniques have various limitations in terms of imaging sensitivity, field of view, flow angle dependence, and imaging depth. In this study, we developed an ultrasound imaging velocimetry approach capable of visualising and quantifying dynamic flow, by combining high-frame-rate plane wave ultrasound imaging, microbubble contrast agents, pulse inversion contrast imaging and speckle image tracking algorithms. The system was initially evaluated in vitro on both straight and carotid-mimicking vessels with steady and pulsatile flows and in vivo in the rabbit aorta. Colour and spectral Doppler measurements were also made. Initial flow mapping results were compared with theoretical prediction and reference Doppler measurements and indicate the potential of the new system as a highly sensitive, accurate, angle-independent and full field-of-view velocity mapping tool capable of tracking and quantifying fast and dynamic flows.     

Leveraging the power of ultrasound for therapeutic design and optimization

Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.     

Use of Contrast-Enhanced Ultrasound in the Assessment of Uterine Fibroids: A Feasibility Study

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.     

微泡造影剂联合高强度聚焦超声治疗兔肝VX-2肿瘤的声像图监测

目的 探讨微泡造影剂联合高强度聚焦超声治疗兔肝VX-2肿瘤时声像图的变化特征及超声监控能力。 方法 45只荷瘤兔随机分为3组，一组接受单纯高强度聚焦超声治疗，另两组为高强度聚焦超声联合微泡造影剂治疗。 结果 高强度聚焦超声联合微泡造影剂治疗肝肿瘤可以大大缩短治疗时间。各治疗组声像图变化的病理学基础各不相同。联合治疗后10min靶区高回声面积与凝固性坏死面积相关性良好（r=0．986，P〈0．05）。 结论 微泡造影剂增强高强度聚焦超声疗效切实可行，联合治疗的声像图特征性变化能够有效判断治疗效果。     

Second Harmonic and Subharmonic for Non-Linear Wideband Contrast Imaging Using a Capacitive Micromachined Ultrasonic Transducer Array

When insonified with suitable ultrasound excitation, contrast microbubbles generate various non-linear scattered components, such as the second harmonic (2H) and the subharmonic (SH). In this study, we exploit the wide frequency bandwidth of capacitive micromachined ultrasonic transducers (CMUTs) to enhance the response from ultrasound contrast agents by selective imaging of both the 2H and SH components simultaneously. To this end, contrast images using the pulse inversion method were recorded with a 64-element CMUT linear array connected to an open scanner. In comparison to imaging at 2H alone, the wideband imaging including both the 2H and SH contributions provided up to 130% and 180% increases in the signal-to-noise and contrast-to-tissue ratios, respectively. The wide-frequency band of CMUTs offers new opportunities for improved ultrasound contrast agent imaging.     

On the Acoustic Properties of Vaporized Submicron Perfluorocarbon Droplets

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.