In Vitro Superharmonic Contrast Imaging Using a Hybrid Dual-Frequency Probe

Superharmonic imaging is an ultrasound contrast imaging technique that differentiates microbubble echoes from tissue through detection of higher-order bubble harmonics in a broad frequency range well above the excitation frequency. Application of superharmonic imaging in three dimensions allows specific visualization of the tissue microvasculature with high resolution and contrast, a technique referred to as acoustic angiography. Because of the need to transmit and receive across a bandwidth that spans up to the fifth harmonic of the fundamental and higher, this imaging approach requires imaging probes comprising dedicated transducers for transmit and receive. In this work, we report on a new dual-frequency probe including two 1.7-MHz rectangular transducers positioned one on each side of a 20-MHz 256-element array. Finite element modeling-based design, fabrication processes and assembly of the transducer are described, as is integration with a high-frequency ultrasound imaging platform. Dual-frequency single-plane-wave imaging was performed with a microbubble contrast agent in flow phantoms and compared with conventional high-frequency B-mode imaging, and resolution and contrast-to-tissue ratio were quantified. This work represents an intermediate but informative step toward the development of dual-frequency imaging probes based on array technology, specifically designed for clinical applications of acoustic angiography.     

Visualization of Microvascular Angiogenesis Using Dual-Frequency Contrast-Enhanced Acoustic Angiography: A Review

Cancerous tumor growth is associated with the development of tortuous, chaotic microvasculature, and this aberrant microvascular morphology can act as a biomarker of malignant disease. Acoustic angiography is a contrast-enhanced ultrasound technique that relies on superharmonic imaging to form high-resolution 3-D maps of the microvasculature. To date, acoustic angiography has been performed with dual-element transducers that can achieve high contrast-to-tissue ratio and resolution in pre-clinical small animal models. In this review, we first describe the development of acoustic angiography, including the principle, transducer design, and optimization of superharmonic imaging techniques. We then detail several preclinical applications of this microvascular imaging method, as well as the current and future development of acoustic angiography as a pre-clinical and clinical diagnostic tool.     

Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging

Currently available microbubbles used for ultrasound imaging and therapeutics are limited to intravascular space due to their size distribution in the micron range. Phase-change contrast agents (PCCAs) have been proposed as a means to overcome this limitation, since droplets formed in the hundred nanometer size range might be able to extravasate through leaky microvasculature, after which they could be activated to form larger highly echogenic microbubbles. Existing PCCAs in the sub-micron size range require substantial acoustic energy to be vaporized, increasing the likelihood of unwanted bioeffects. Thus, there exists a need for PCCAs with reduced acoustic activation energies for use in imaging studies. In this article, it is shown that decafluorobutane, which is normally a gas at room temperature, can be incorporated into metastable liquid sub-micron droplets with appropriate encapsulation methods. The resulting droplets are activatable with substantially less energy than other favored PCCA compounds. Decafluorobutane nanodroplets may present a new means to safely extend ultrasound imaging beyond the vascular space.     

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。     

Amplitude-Modulation Chirp Imaging for Contrast Detection

We propose an amplitude-modulation chirp imaging method for contrast detection with high-frequency ultrasound. Our proposed method detects microbubbles by extracting and then selectively compressing the component of the backscattered chirp signal modulated by changes in the radii of microbubbles at their resonance frequency. Microbubbles are sonicated simultaneously with a narrowband, low-frequency pumping signal at their resonance frequency and a wideband, high-frequency imaging chirp signal. Changes in the radii of the resonant microbubbles result in periodic changes in their acoustic cross section that modulate the amplitude of the backscattered imaging chirp signal, forming pumping and imaging frequency sum-and-difference chirp terms. The frequency-sum or -difference chirp component is then extracted by a bandpass filter (BPF). Because a long imaging pulse duration is required to obtain a sufficient modulation depth on the chirp for contrast detection and to facilitate frequency-sum-and-difference signal extraction with the BPF, a chirp with a longer-than-usual waveform is used so pulse compression of the extracted chirp signal can then be performed to maintain the axial resolution, and even further improve the signal-to-noise ratio and contrast-to-tissue ratio. Experiments performed on flow phantoms with and without a speckle-generating background were performed to demonstrate the efficacy of the proposed technique. These results indicate that our proposed method can potentially provide high-resolution contrast detection in the microvasculature. (E-mail: ckyeh@mx.nthu.edu.tw)     

多模态超声造影剂的现状及研究进展

近年来,随着多种医学成像技术的相互融合和分子影像技术的迅速发展,以超声分子显像为基础,同时具有多种影像学对比显影能力的多模态超声造影剂已成为当前超声影像学领域的研究热点之一。多模态超声造影剂不仅可以提高超声成像的分辨力、敏感性、对比度,还能在一定程度改善传统单一影像诊断的局限性,实现多种方式成像的优势互补,具有广阔的应用前景。本文就多模态超声造影剂的现状及进展进行综述。     

微泡造影剂在肝脏疾病诊治中的研究进展

新型造影剂的发展,使肝脏的超声造影正发生巨大的变化.当静脉注射不同类型的超声造影剂时,微泡通过肺循环,在特定的影像程序下,不仅可以增强多普勒信号、改善灰阶超声图像的质量,而且可以实时地观察微血管及组织的血流灌注.通过在微泡上偶联肿瘤特异性抗体等方法使微泡具有靶向功能,大大提高了对肿瘤诊断的准确性,同时还可以携带药物与基因用于肿瘤的治疗.随着靶向超声微泡技术的发展,靶向超声微泡必将为超声显像与临床治疗提供一种有效的方法.     

Microbubble contrast for radiological imaging: 1. Principles

Microbubble contrast for ultrasound imaging in radiology has finally come of age, adding entirely new capabilities to real time imaging. Following a bolus injection into a peripheral vein of as little as 0.1mL of an aqueous suspension, contrast specific imaging modes show parenchymal and lesional perfusion in real time in the major organs of the abdomen and pelvis as well as breast, thyroid and prostate. These new imaging modes exploit the unique interaction between ultrasound and microbubbles, which gives rise to nonlinear echoes that are readily distinguishable from those of tissue. Furthermore, microbubbles can be deliberately disrupted by the ultrasound imaging field. The rate at which fresh bubbles then replenish the scanplane can be metered in subsequent images, offering a means to quantify both flowrate and relative vascular volume of the microvasculature in both organs and solid lesions.     

Imaging Microvasculature with Contrast-Enhanced Ultraharmonic Ultrasound

Atherosclerotic plaque neovascularization was shown to be one of the strongest predictors of future cardiovascular events. Yet, the clinical tools for coronary wall microvasculature detection in vivo are lacking. Here we report an ultrasound pulse sequence capable of detecting microvasculature invisible in conventional intracoronary imaging. The method combines intravascular ultrasound with an ultrasound contrast agent, i.e., a suspension of microscopic vascular acoustic resonators that are small enough to penetrate the capillary bed after intravenous administration. The pulse sequence relies on brief chirp excitations to extract ultraharmonic echoes specific to the ultrasound contrast agent. We implemented the pulse sequence on an intravascular ultrasound probe and successfully imaged the microvasculature of a 6 days old chicken embryo respiratory organ. The feasibility of microvasculature imaging with intravascular ultrasound sets the stage for a translation of the method to studies of intra-plaque neovascularization detection in humans.     

超声造影在肝脏疾病诊断和治疗中的应用

新型造影剂和超声造影技术的发展,使肝脏的超声影像正在发生巨大的变化.当静脉注射不同类型的超声造影剂时,微泡通过肺循环,在特定的影像程序下,不仅可以增强多普勒信号、改善灰阶超声图像的质量,而且可以实时的观察微血管及组织的血流灌注.在肝脏疾病的诊断和治疗中可提供至关重要的信息,而这在常规超声是完全不可能的.对超声造影在肝脏疾病诊断和治疗中的应用综述.     

包裹Gd-DTPA的高分子材料超声造影剂显影效果兔实验研究

目的 观察自制包裹Gd-DTPA的高分子材料超声造影剂(Gd-PLGA)的显影效果、特点及其安全性.方法 质量浓度为10%的Gd-PLGA溶液,按0.5 ml/kg剂量经兔耳缘静脉团注,分别对肝血管、肝实质进行超声及磁共振成像,并对肝实质信号强度进行动态定量分析;所有兔在Gd-PLGA注射之前以及注射之后7、14、30 d静脉取血进行肝功能和肾功能检测.结果 Gd-PLGA在超声显像中能明显增强肝血管、肝实质回声强度,而在磁共振成像中肝实质信号强度亦较造影前增强,作用时间均持续约30 min;所有兔在实验过程中以及实验后观察1个月均未发现明显异常,且各组肝、肾功能指标差异无统计学意义.结论 Gd-PLGA是一安全、显影效果好、持续时间长、能实现实时超声及磁共振双重造影的新型多功能高分子材料超声造影剂.     

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（1MHz）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。     

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(1MHz)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。     

In Vivo Molecular Imaging Using Low-Boiling-Point Phase-Change Contrast Agents: A Proof of Concept Study

Sub-micron phase-change contrast agents (PCCAs) have been proposed as a tool for ultrasound molecular imaging based on their potential to extravasate and target extravascular markers and also because of the potential to image these contrast agents with a high contrast-to-tissue ratio. We compare in vivo ultrasound molecular imaging with targeted low-boiling-point PCCAs and targeted microbubble contrast agents. Both agents were targeted to the intravascular (endothelial) integrin α_vß₃via a cyclic RGD peptide (cyclo-Arg–Gly–Asp–D-Tyr–Cys) mechanism and imaged in vivo in a rodent fibrosarcoma model, which exhibits angiogenic microvasculature. Signal intensity was measured using two different techniques, conventional contrast-specific imaging (amplitude/phase modulation) and a droplet vaporization imaging sequence, which detects the unique signature of vaporizing PCCAs. Data indicate that PCCA-specific imaging is more sensitive to small numbers of bound agents than conventional contrast imaging. However, data also revealed that contrast from targeted microbubbles was greater than that provided by PCCAs. Both control and targeted PCCAs were observed to be retained in tissue post-vaporization, which was expected for targeted agents but not expected for control agents. The exact mechanism underlying this observation remains unknown.     

自制脂质类超声造影剂肾增强效果的实验研究

目的探讨自制脂质类超声造影剂对正常兔肾的增强效果,以及不同显像方式、造影剂量对造影效果的影响与变化规律.方法 5只健康新西兰大白兔经耳缘静脉注射不同剂量的自制脂质类超声造影剂,剂量分别为0.01、0.03、0.05、0.08、0.10、0.13、0.15 ml/kg,每只兔各剂量均注射2次.用基波显像(FI)和实时造影匹配成像(CnTi)两种显像方式观察肾实质造影效果.结果 7种剂量水平均可使肾实质回声增强.CnTi随造影剂量增加肾实质增强强度提高,且增强时间延长.FI在上述各剂量水平肾实质峰值视频强度及增强时间均显著低于CnTi(P＜0.001).结论自制脂质类超声造影剂能够实现显著的肾实质增强效果,剂量-效应关系在CnTi造影中变化敏感,在FI造影中变化不敏感.     

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.     

Nanoparticle‐stabilized microbubbles for multimodal imaging and drug delivery

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)‐coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self‐assembly of NPs at the MB air–liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP–MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs. Copyright © 2015 John Wiley & Sons, Ltd.     

In Vivo Imaging of Microvasculature during Anesthesia with High-Resolution Photoacoustic Microscopy

Anesthesia monitoring is extremely important in improving the quality of anesthesia and ensuring the safety of patients in operation. Photoacoustic microscopy (PAM) is proposed to in vivo image the skin microvasculature of 10 nude mice undergoing general anesthesia by using the isoflurane gas with a concentration of 3%. Benefiting from strong optical absorption of hemoglobin, PAM has good contrast and high resolution in mapping of microvasculature. A series of high quality images can clearly reveal the subtle changes of capillaries in morphology over time. Two indices, vessel intensity and vessel density, are extracted from these images to measure the microvasculature quantitatively. The imaging results show that the vessel intensity and density are increased over time. After 65?min, the vessel intensity increased 42.7?±?8.6% and the density increased 28.6?±?12.2%. These indices extracted from photoacoustic images accurately reflect the greater blood perfusion undergoing general anesthesia. Additionally, abnormal reductions of vessel intensity and density are also observed as overtime anesthesia. This preclinical study suggests that PAM holds potential to monitor anesthesia by imaging the skin microvasculature.     

Effect of A-scan rate and interscan interval on optical coherence angiography

Optical coherence tomography angiography (OCTA) can provide rapid, volumetric, and noninvasive imaging of tissue microvasculature without the requirement of exogenous contrast agents. To investigate how A-scan rate and interscan time affected the contrast and dynamic range of OCTA, we developed a 1.06-µm swept-source OCT system enabling 100-kHz or 200-kHz OCT using two light sources. After system settings were carefully adjusted, almost the same detection sensitivity was achieved between the 100-kHz and 200-kHz modalities. OCTA of ear skin was performed on five mice. We used the variable interscan time analysis algorithm (VISTA) and the designated scanning protocol with OCTA images reconstructed through the correlation mapping method. With a relatively long interscan time (e.g., 12.5 ms vs. 6.25 ms for 200-kHz OCT), OCTA can identify more intricate microvascular networks. OCTA image sets with the same interscan time (e.g., 12.5 ms) were compared. OCTA images acquired with a 100-kHz A-scan rate showed finer microvasculature than did other imaging modalities. We performed quantitative analysis on the contrast from OCTA images reconstructed with different A-scan rates and interscan time intervals in terms of vessel area, total vessel length, and junction density.     

Microbubble Enzyme-Linked Immunosorbent Assay for the Detection of Targeted Microbubbles in in Vitro Static Binding Assays

Targeted microbubbles (MBs) are ultrasound contrast agents that are functionalized with a ligand for ultrasound molecular imaging of endothelial markers. Novel targeted MBs are characterized in vitro by incubation in protein-coated wells, followed by binding quantification by microscopy or ultrasound imaging. Both methods provide operator-dependent results: Between 3 and 20 fields of view from a heterogeneous sample are typically selected for analysis by microscopy, and in ultrasound imaging, different acoustic settings affect signal intensities. This study proposes a new method to reproducibly quantify MB binding based on enzyme-linked immunosorbent assay (ELISA), in which bound MBs are revealed with an enzyme-linked antibody. MB-ELISA was adapted to in vitro static binding assays, incubating the MBs in inverted position or by agitation, and compared with microscopy. The specificity and sensitivity of MB-ELISA enable the reliable quantification of MB binding in a rapid, high-throughput and whole-well analysis, facilitating the characterization of new targeted contrast agents.