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.     

Applications of ultrasmall superparamagnetic iron oxide contrast agents in the MR study of animal models

Ultrasmall superparamagnetic iron oxide nanoparticles have been widely used during the past decade as MR intravascular contrast agents in the study of animal models. Such agents enhance both T1 and T2/T2* relaxation, although for animal studies it is the later type of enhancement that is most commonly exploited. Their strong microscopic intravascular susceptibility effect enables the local blood volume distribution to be mapped in various organs. High spatial resolution and sensitivity can be achieved, because the long half-life of these agents in blood, combined with anesthetization, permits steady-state measurements over extended periods. This capability has been utilized to study the cerebrovascular blood volume distributions and their changes in normal, activated, pathologic and pharmacologically or genetically modified states, particularly in rodent animal models. It has also been applied to study blood volume changes in other tissues, such as the myocardium. The relaxation rate shifts Delta R2 and Delta R2* induced by iron oxide agents may differ depending on certain morphological characteristics of the microvascular network, and sensitive Delta R2 and Delta R2* mapping can potentially provide, in addition to blood volume, measurement of other important microvascular parameters such as blood vessel density and size. This work aims to review the applications of ultrasmall superparamagnetic iron oxide contrast agents in MR animal studies, with an emphasis on the investigation of microvascular parameters.     

Understanding the limitations of ultrasonic backscatter measurements from microbubble populations

Despite over ten years of in vitro investigations of ultrasound contrast agents, the level of understanding of their behaviour in ultrasound fields is limited. Several problems associated with these investigations, particular to the nature of contrast agents, are discussed. Using a commercial scanner the RF normalized backscatter of two different contrast agents (Definity and Quantison) was measured at different suspension concentrations and acoustic pressures. Both contrast agents scattered ultrasound nonlinearly and the backscatter showed a dependence on acoustic pressure. In order to assess the average behaviour of the agents across the range of acoustic pressures and microbubble concentrations the experimental data were fitted to a theoretically acceptable model using nonlinear regression analysis. The analysis showed that both the backscatter and the attenuation of the Quantison suspensions displayed a higher order of dependence on acoustic pressure than the Definity suspensions. It was also discovered that Quantison microbubbles did not demonstrate uniform behaviour across the acoustic pressure range. At lower acoustic pressures the behaviour could not follow a model similar to that which predicted the behaviour at higher acoustic pressures, which was mainly due to the fact that free bubbles were released in a fashion dependent on acoustic pressure. The fact that two different populations of scatterers exist in the same suspensions makes the assessment of the behaviour of the particular agent impossible with the high concentrations that are commonly used. Very low concentration suspensions whereby single scattering events can be monitored should be more useful. In conclusion, the approach of using high microbubble concentrations in order to investigate the properties of ultrasonic contrast agents is limited in that the results of such studies cannot be used to understand the behaviour of single 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.     

Lipid-based nanoparticles for contrast-enhanced MRI and molecular imaging

In the field of MR imaging and especially in the emerging field of cellular and molecular MR imaging, flexible strategies to synthesize contrast agents that can be manipulated in terms of size and composition and that can be easily conjugated with targeting ligands are required. Furthermore, the relaxivity of the contrast agents, especially for molecular imaging applications, should be very high to deal with the low sensitivity of MRI. Lipid-based nanoparticles, such as liposomes or micelles, have been used extensively in recent decades as drug carrier vehicles. A relatively new and promising application of lipidic nanoparticles is their use as multimodal MR contrast agents. Lipids are amphiphilic molecules with both a hydrophobic and a hydrophilic part, which spontaneously assemble into aggregates in an aqueous environment. In these aggregates, the amphiphiles are arranged such that the hydrophobic parts cluster together and the hydrophilic parts face the water. In the low concentration regime, a wide variety of structures can be formed, ranging from spherical micelles to disks or liposomes. Furthermore, a monolayer of lipids can serve as a shell to enclose a hydrophobic core. Hydrophobic iron oxide particles, quantum dots or perfluorocarbon emulsions can be solubilized using this approach. MR-detectable and fluorescent amphiphilic molecules can easily be incorporated in lipidic nanoparticles. Furthermore, targeting ligands can be conjugated to lipidic particles by incorporating lipids with a functional moiety to allow a specific interaction with molecular markers and to achieve accumulation of the particles at disease sites. In this review, an overview of different lipidic nanoparticles for use in MRI is given, with the main emphasis on Gd-based contrast agents. The mechanisms of particle formation, conjugation strategies and applications in the field of contrast-enhanced, cellular and molecular MRI are discussed.     

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.     

Contrast-enhanced MRI of murine myocardial infarction - part II

Mouse models are increasingly used to study the pathophysiology of myocardial infarction in vivo. In this area, MRI has become the gold standard imaging modality, because it combines high spatial and temporal resolution functional imaging with a large variety of methods to generate soft tissue contrast. In addition, (target-specific) MRI contrast agents can be employed to visualize different processes in the cascade of events following myocardial infarction. Here, the MRI sequence has a decisive role in the detection sensitivity of a contrast agent. However, a straightforward translation of clinically available protocols for human cardiac imaging to mice is not feasible, because of the small size of the mouse heart and its extremely high heart rate. This has stimulated intense research in the development of cardiac MRI protocols specifically tuned to the mouse with regard to timing parameters, acquisition strategies, and ECG- and respiratory-triggering methods to find an optimal trade-off between sensitivity, scan time, and image quality. In this review, a detailed analysis is given of the pros and cons of different mouse cardiac MR imaging methodologies and their application in contrast-enhanced MRI of myocardial infarction.     

高分子材料超声对比剂的制备及显影特征

背景：目前所用的超声对比剂均为内含不同气体成分的微气泡,其外壳材料多数为表面活性剂类、人血蛋白质类、脂类等。随着高分子化学的发展,高分子材料对比剂成为超声对比剂研究领域的热点。 目的：探讨各种超声对比剂制备中遇到的困难以及解决方法,从而最终寻找合理的高分子材料超声对比剂。 方法：采用电子检索的方式,在万方数据库(http://www.wanfangdata.com.cn/)中检索2005-01/2010-12有关高分子材料应用于超声造影方面的研究文章,关键词为“高分子材料,超声,对比剂”。排除重复研究、普通综述或Meta分析类文章,筛选纳入26篇文献进行评价。 结果与结论：近年来随着高分子科学与多学科融合的分子医学的兴起和快速发展,显像对比剂受到了越来越广泛的关注。高分子材料超声对比剂由于具有好的生物相容性,粒径大小均匀,良好的抗压性能,较长的显影持续时间等特点,已成为目前研究的热点。其中靶向微泡对比剂经静脉注射可到达特定靶区,低功率超声作用下可提高局部组织显影的分辨率。携带治疗药物或基因的靶向微泡对比剂在低频(1 MHz)超声作用下可以产生瞬态空化效应,迫使细胞膜的通透性增加,从而有效提高了药物或基因的转染率。如今,靶向微泡携抗肿瘤药物联合超声作用正逐渐成为治疗肿瘤的一种新模式,是近期医学研究的一个热点。超声联合靶向微泡技术在临床诊断和治疗中显示出了较大的优势,但其准确的生物学机制目前医学界还未清楚,超声治疗参数需进一步优化。     

Understanding the fundamentals of perfluorocarbons and perfluorocarbon emulsions relevant to in vivo oxygen delivery

The unique behavior of perfluorocarbons (PFCs), including their high oxygen dissolving capacity, hydrophobic and lipophobic character, and extreme inertness, derive directly, in a predictable manner, from the electronic structure and spatial requirements of the fluorine atom. Their low water solubility is key to the prolonged in vivo persistence of the now commercially available injectable microbubbles that serve as contrast agents for diagnostic ultrasound imaging. Oxygent, a stable, small-sized emulsion of a slightly lipophilic, rapidly excreted PFC, perfluorooctyl bromide (perflubron), has been engineered. Significant oxygen delivery has been established in animal models and through Phase II and III human clinical trials. However, an inappropriate testing protocol and the lack of funding led to temporary suspension of the trials.     

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.     

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.     

Ultrasonic characterization of ultrasound contrast agents

The main constituent of an ultrasound contrast agent (UCA) is gas-filled microbubbles. An average UCA contains billions per ml. These microbubbles are excellent ultrasound scatterers due to their high compressibility. In an ultrasound field they act as resonant systems, resulting in harmonic energy in the backscattered ultrasound signal, such as energy at the subharmonic, ultraharmonic and higher harmonic frequencies. This harmonic energy is exploited for contrast enhanced imaging to discriminate the contrast agent from surrounding tissue. The amount of harmonic energy that the contrast agent bubbles generate depends on the bubble characteristics in combination with the ultrasound field applied. This paper summarizes different strategies to characterize the UCAs. These strategies can be divided into acoustic and optical methods, which focus on the linear or nonlinear responses of the contrast agent bubbles. In addition, the characteristics of individual bubbles can be determined or the bubbles can be examined when they are part of a population. Recently, especially optical methods have proven their value to study individual bubbles. This paper concludes by showing some examples of optically observed typical behavior of contrast bubbles in ultrasound fields.     

Model-based ultrasound tomography: tissue phantom experiments

We present a detailed experimental study to evaluate our finite element based nonlinear reconstruction algorithm for recovery of acoustic properties in heterogeneous scattering media. Using a circularly scanning ultrasound system at 500 KHz, tissue phantom experiments were performed to study spatial resolution and contrast issues in model-based ultrasound tomography. Our results show that both acoustic attenuation and speed images can be quantitatively reconstructed in terms of the location, size, shape, and acoustic property value of the target when different contrast levels between the target and background were used. We also demonstrate that a high contrast target as small as 3 mm in diameter can be quantitatively resolved with our acoustic speed and attenuation images.     

Patient dose measurements in photon fields by means of silicon semiconductor detectors

Semiconductor detectors based on p-type silicon and designed for in vivo measurement of entrance dose at the reference point from photon radiation fields, are described. To estimate the absorbed dose at the reference point from measurements with a thin detector, field-size dependent correction factors must be applied to the reading, as the shape of the dose buildup curve varies with field size. To decrease or avoid field-size dependent correction factors, the detector can be covered with a buildup cap. The presence of such a detector will cause perturbation of the radiation field. Therefore, the design of a detector, irrespective of its type, intended for patient dosimetry involves a compromise between minimizing the radiation field perturbation and minimizing field-size dependent correction factors. Detectors with three different buildup caps were designed to cover the energy range from cobalt-60 to 16-MV x rays. The three different detector types were investigated with respect to their signal dependence on field size, field perturbation, and directional dependence. A summary of radiation damage effects on sensitivity, and of sensitivity variation with temperature is also presented.     

组织声学特性对高强度聚焦超声温度场的影响

目的 数值仿真组织声学特性对高强度聚焦超声（HIFU）焦域处温度场的影响,为HIFU治疗安全性和可靠性提供理论依据.方法 以实测新鲜离体猪肝组织不同温度下的声速和衰减系数为依据,利用时域有限差分（FDTD）法数值仿真研究HIFU治疗过程中组织内声速、衰减系数的变化和温度场的分布,分析讨论声速和衰减系数变化对60 ℃以上可治疗区域大小、位置的影响.结果 随着照射时间的延长,焦域处肝组织温升增大,声速下降,声衰减系数增大.随着声速的变化,形成的可治疗区域变大,焦点位置向远离换能器方向移动;随着声衰减系数的变化,焦域大小和焦点位置几乎不变.结论 猪肝组织内声速的变化对可治疗焦域的位置和大小影响较大;声衰减系数的变化对焦域的影响较小.     

Developed γM haemolytic plaque-forming cells in chickens

Incorporation of rabbit anti-μ serum into agarose gels inhibited direct anti-SRBC PFCs using chicken complement, but developed equal numbers of indirect PFCs binding guinea-pig complement. The majority of attempts to demonstrate direct PFCs against HSA were unsuccessful, although low numbers of barely discernible PFCs were obtained by using HSA-SRBC sensitized with carbodiimide at room temperature; indirect PFCs were developed, however, by the anti-μ serum. During the primary response, developed anti-HSA PFCs could not be assigned to IgM or IgG classes using reduction and alkylation. Concanavalin A completely inhibited direct anti-SRBC and SIII PFCs, although developed γM PFCs were only inhibited partially. The identity of direct and anti-μ developed PFCs against SIII was demonstrated by the replica technique. None of the approaches applied provided any support for the existence of structural heterogeneity of IgM within the PFC population.     

Preparation and evaluation of poly(L-lactide-co-glycolide) (PLGA) microbubbles as a contrast agent for myocardial contrast echocardiography

A kind of absorbable PLGA microbubble-based contrast agent (PLGA microspheres with porous or hollow inner structure) was fabricated by an improved double emulsion-solvent evaporation method. The contrast efficiency was evaluated and proved both in vitro and in vivo. By adjusting the polymer concentration and volume of the inner aqueous phase during the fabrication of microbubbles, the inner structure of the microbubbles could be controlled. Both air-filled and perfluoropropane-filled microbubbles can opacify the left ventricle. However, when compared with air-filled microbubbles, perfluoropropane-filled microbubbles can produce significantly longer enhancement in left ventricle in the dog model due to the lower diffusivity and lower solubility of perfluoropropane in blood. A suspension of perfluoropropane-filled PLGA microbubbles (1.8 microm average microbubbles size, 2 x 10(8) microbubbles/mL concentration) has successfully and safely achieved myocardial opacification in closed-chest dogs. A perfusion defect was observed in both of the two dogs with acute myocardial infarction with Power Contrast Imaging (PCI) triggered technology. In the examination of contrast in both ventricular and myocardial opacification, the high mechanical index (MI) was found to have superior contrast sensitivity over the low MI for PLGA-based contrast agents.     

水听器式便携超声参数测量系统的研制

目的：开发了一套测量声输出参数的系统，为进一步研制适用于军事计量巡检工作的便携式仪器奠定基础。方法：采用水听器作为换能器，采集超声信号存空间声场焦平面上一点的声压波形。通过对声压波形的参数进行数值运算，得到所关心的超声声输山参数。结果：能够在误差允许的范围内测量出主要的声输出参数。结论：该方法切实可行，得到的数据可重复性好。     

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。     

Single-layer and dual-layer contrast-enhanced mammography using amorphous selenium flat panel detectors

The accumulation of injected contrast agents allows the image enhancement of lesions through the use of contrast-enhanced mammography. In this technique, the combination of two acquired images is used to create an enhanced image. There exist several methods to acquire the images to be combined, which include dual energy subtraction using a single detection layer that suffers from motion artifacts due to patient motion between image acquisition. To mitigate motion artifacts, a detector composed of two layers may be used to simultaneously acquire the low and high energy images. In this work, we evaluate both of these methods using amorphous selenium as the detection material to find the system parameters (tube voltage, filtration, photoconductor thickness and relative intensity ratio) leading to the optimal performance. We then compare the performance of the two detectors under the variation of contrast agent concentration, tumor size and dose. The detectability was found to be most comparable at the lower end of the evaluated factors. The single-layer detector not only led to better contrast, due to its greater spectral separation capabilities, but also had lower quantum noise. The single-layer detector was found to have a greater detectability by a factor of 2.4 for a 2.5 mm radius tumor having a contrast agent concentration of 1.5 mg ml(-1) in a 4.5 cm thick 50% glandular breast. The inclusion of motion artifacts in the comparison is part of ongoing research efforts.