超声波声孔效应中气泡动力学的研究

在超声快速制取组织细胞病理切片的过程中，发现激励信号对切片制取效果有明显的影响.为了掌握超声激励信号对组织细胞的影响规律，达到快速制取病理切片的最佳状态，从气泡空化模型入手，通过改变激励信号频率、声压、气泡初始半径和液体黏滞系数等参量，研究了声孔效应中气泡动力学激励机制.数值计算表明：空化泡振动随激励声压增强而升高，随液体黏滞系数增强而减弱；一定频率范围内空化泡振动能保持在膨胀、收缩和振荡的稳定空化状态，存在空化泡稳态振动的最佳激励频率；一定初始半径能保证空化泡产生稳定的振动，存在空化泡稳态振动幅度最大的初始半径.实际操作中，在频率、声压、初始半径和黏滞系数综合作用的若干空化阈内，声孔效应使超声快速法制取细胞组织切片获得最佳效果. 关键词： 声孔效应 超声空化 气泡振动 稳态空化域     

振动声调制技术下微裂纹的非线性响应分析

采用非线性弹簧模型对振动声调制技术下微裂纹产生的非线性调制效应进行解释。从理论上分析了振动声调制技术下微裂纹产生非线性调制现象的原理,将微裂纹的开合状态等效为非线性弹簧。利用有限元软件构建并仿真微裂纹非线性弹簧模型,分别观察不同激励下裂纹界面的开合状态及混合声波的传播特性。结果表明,微裂纹界面随低频声波呈周期性开合,使得通过裂纹界面的高频声波与低频声波相互调制而产生非线性现象。在以上研究基础上,开展振动声调制实验研究。对比仿真结果,非线性调制系数R在实验条件下和仿真随低频激励幅值的变化趋势相同,证明了该模型的正确性。该模型为振动声调制技术应用于微裂纹的超声非线性检测提供了理论依据。      

基于微泡共振的快速微流体声学混合方法研究

微流体在生物医学、化学工程等领域应用广泛,并具有重大意义.在预处理中,液体混合也是关键且最为必要的前序.为了提高微流控腔道内液体混合的效率,本文提出基于单微泡振动的声学混合器,通过微泡共振,产生声微流,声微流形成的剪切力将在流体中产生微扰动,实现液体的混合.设计了底面直径为40μm的微孔结构,由于液体表面张力作用形成微泡,在共振频率为165 kHz的压电换能器激励下,气泡发生共振产生声微流.通过对压电换能器输入不同能量,获取混合液体的最优参数,可在37.5 ms内实现混合效果,混合均匀度达到92.7%.本文设计的单微泡振动混合器结构简单、混合效率高、混合时间短、输入能量低,可为生物化学等方面的研究提供强有力的技术支撑.     

微圆孔非线性声阻抗及声整流现象的实验研究

在高声强下测量了微圆孔处声激发射流的速度和微圆孔的非线性声阻抗。随声压级的增加声激发射流的速度增大,实验中射流速度在 ０－１９ｍ／ｓ范围内变化,这表明出现一种强烈声整流现象;与此同时微圆孔声阻明显增大,而声抗减小,声抗最小值约是其线性值的０．７倍。此外实验结果还验证了一种微园孔声学非线性效应离散涡模型的合理性。     

气泡幕的声透射损失和对水中壳体减振的实验研究

通过不同放气孔径和不同放气压差所形成的气泡幕的透声实验,获得了含气浓度在大范围内变化的气泡幕透声损失与频率关系的实验资料。在气泡幕对浮于水中壳体振动的影响的实验中,得到了气泡幕对壳体振动有明显抑制作用的实验结果。     

多层膜磁性微泡的非线性声振动特性

超顺磁性氧化铁纳米粒子与造影剂微泡结合形成磁性微泡,用于产生多模态造影剂,以增强医学超声和磁共振成像.将装载有纳米磁性颗粒的微泡包膜层看作由磁流体膜与磷脂膜组合而成的双层膜结构,同时考虑磁性纳米颗粒体积分数a对膜密度及黏度的影响,从气泡动力学基本理论出发,构建多层膜结构磁性微泡非线性动力学方程.数值分析了驱动声压和频率等声场参数、颗粒体积分数、膜层厚度以及表面张力等膜壳参数对微泡声动力学行为的影响.结果表明,当磁性颗粒体积分数较小且a≤0.1时,磁性微泡声响应特性与普通包膜微泡相似,微泡的声频响应与其初始尺寸和驱动压有关;当驱动声场频率f为磁性微泡共振频率f0的2倍(f=2f0)时,微泡振动失稳临界声压最低;磁性颗粒的存在抑制了泡的膨胀和收缩但抑制效果非常有限;磁性微泡外膜层材料的表面张力参数K及膜层厚度d也会影响微泡的振动,当表面张力参数及膜厚取值分别为0.2—0.4 N/m及50—150 nm时,可观察到气泡存在不稳定振动响应区.     

含气泡液体中声传播的解析解及其强非线性声特性

声波在含气泡的液体中传播时,气泡的受迫振动会引起强的声散射,并且由于振动的非线性,使得气泡产生的次级波不仅含有基频成分,而且还会有高次谐波。本文从理论上描述了气泡个数随尺并给出了含气泡液体的等效非线性声参数Ｂ／Ａ的计算公式理论与已有的实验观测符合较好,文中对含气泡水的声速和声衰减等特性也进行了讨论。     

H-22细胞声孔效应的应力阈值

理论和实验研究了超声空化场中的H-22型肝癌细胞产生可逆声孔效应的剪应力阈值.本文用1.37 MHz的聚焦声场,当超顺磁性纳米氧化铁在细胞悬液中的终浓度为410 μg/mL,换能器负载电功率为2 W,超声辐照60 s,细胞存活率90%以上时,有45.9±13.5%的细胞显示普鲁士蓝染阳性,暗示超声作用下,这些细胞表面曾出现可逆性微孔而使磁性微粒由此进入细胞内.利用无界自由空间微泡运动方程的球对称稳态解对实验条件下细胞膜表面的切变应力进行数值估算,结果表明,使H-22细胞产生可逆性声孔效应的微流剪应力阈值为697 Pa. 关键词： 声孔效应 磁性标记 微流 剪应力     

超声造影剂微气泡的包膜黏弹特性的定量表征研究

包膜黏弹特性显著影响微气泡超声造影剂的诊断及治疗应用效果. 本文结合原子力显微镜技术及声衰减特性测量提出了一种对微气泡造影剂包膜黏弹特性定量表征的新方法. 首先采用原子力显微镜技术进行机械特性分析得到包膜微气泡的有效硬度及体弹性模量; 然后测量声衰减特性, 基于微气泡动力学理论, 计算包膜微气泡的体黏度系数. 为验证方法的有效性, 实验制备了直径为1-5 μm的白蛋白包膜微气泡造影剂, 原子力显微镜测量的有效硬度和体弹性模量分别为0.149±0.012 N/m和8.31±0.667 MPa, 并与粒径无关. 声衰减特性测量和动力学理论拟合的包膜微气泡的体黏度系数为0.374±0.003 Pa·s. 该方法可推广至其他种类包膜微气泡的黏弹特性表征, 对超声造影剂的制备及其诊断和治疗应用有积极意义.     

弹性管中泡群内气泡的非线性声响应

将弹性管壁视为膜弹性结构,得到了管径较大弹性管中泡群内气泡弱非线性振动的动力学模型.利用逐级近似法对气泡的非线性共振频率、基频振动响应特性进行了理论分析.结果表明:气泡共振频率主要受泡群内气泡间相互作用的影响;气泡的非线性共振频率将发生偏移,其偏移量取决于共振响应振幅;气泡的声响应区存在最大频率值;在声响应的高频率区内声响应幅值有多值性.     

Effects of extracellular matrix rigidity on sonoporation facilitated by targeted microbubbles: Bubble attachment,bubble dynamics,and cell membrane permeabilization

In this study, we investigated the effects of extracellular matrix rigidity, an important physical property of microenvironments regulating cell morphology and functions, on sonoporation facilitated by targeted microbubbles, highlighting the role of microbubbles. We conducted mechanistic studies at the cellular level on physiologically relevant soft and rigid substrates. By developing a unique imaging strategy, we first resolved details of the 3D attachment configurations between targeted microbubbles and cell membrane. High-speed video microscopy then unveiled bubble dynamics driven by a single ultrasound pulse. Finally, we evaluated the cell membrane permeabilization using a small molecule model drug. Our results demonstrate that: (1) stronger targeted microbubble attachment was formed for cells cultured on the rigid substrate, while six different attachment configurations were revealed in total; (2) more violent bubble oscillation was observed for cells cultured on the rigid substrate, while one third of bubbles attached to cells on the soft substrate exhibited deformation shortly after ultrasound was turned off; (3) higher acoustic pressure was needed to permeabilize the cell membrane for cells on the soft substrate, while under the same ultrasound condition, acoustically-activated microbubbles generated larger pores as compared to cells cultured on the soft substrate. The current findings provide new insights to understand the underlying mechanisms of sonoporation in a physiologically relevant context and may be useful for the clinical translation of sonoporation.     

Development of a theoretical model describing sonoporation activity of cells exposed to ultrasound in the presence of contrast agents

Sonoporation uses ultrasound, with the aid of ultrasound contrast agents (UCAs), to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds noninvasively into specific target cells. The objective of this study was to determine if a computational model describing shear stress on a cell membrane due to microstreaming would successfully reflect sonoporation activity with respect to the peak rarefactional pressure. The theoretical models were compared to the sonoporation results from Chinese hamster ovary cells using Definity(?) at 0.9, 3.15, and 5.6 MHz and were found to accurately describe the maximum sonoporation activity, the pressure where a decrease in sonoporation activity occurs, and relative differences between maximum activity and the activity after that decrease. Therefore, the model supports the experimental findings that shear stress on cell membranes secondary to oscillating UCAs results in sonoporation.     

Radionuclide tumour therapy with ultrasound contrast microbubbles

Radionuclides have shown to be effective in tumour therapy. However, the side effects determine the maximum deliverable dose. Recently, it has been demonstrated that cells can be permeabilised through sonoporation using ultrasound and contrast microbubbles. The use of sonoporation in treatment of tumours may increase the anti-tumour efficacy of radionuclide treatment. The mechanisms as well as the effects sonoporation in tumour treatment strategies are still not understood. The purpose of this study is to determine the effects of ultrasound and contrast microbubbles on the internalisation of the radionuclide (111)In-DOTA-Tyr(3)-octreotate in tumour cells. To optimize ultrasound settings for ultrasound adjunctive tumour therapy we incubated rat pancreatic CA20948 tumour cells with two dyes (MW 40 and 70 kDa). The uptake levels were compared with cells treated with ultrasound and contrast microbubbles for different ultrasound settings. The highest molecular uptake was found with addition of contrast microbubbles (ratio of 10 bubbles to 1 cell) and with the ultrasound setting: duty cycle 0.013%, mechanical index (MI) 0.42, and treatment times of 30 and 60 min. These settings were used to enhance the internalisation of (111)In-DOTA-Tyr(3)-octreotate. We found a 160% higher internalisation of (111)In-DOTA-Tyr(3)-octreotate by tumour cells adjunctively treated with ultrasound and contrast microbubbles compared to untreated cells. These results show that adjunctive tumour treatment with the radionuclide (111)In-DOTA-Tyr(3)-octreotate and ultrasound contrast microbubbles may be feasible. When using adjunctive ultrasound contrast microbubble treatment, a lower radionuclide doses are required to reach the same anti-tumour effect.     

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.     

超声造影剂微泡非线性动力学响应的机理及相关应用

随着生命科学及现代医学的发展, 一体化无创精准诊疗已经日益成为人们关注的焦点问题, 而关于超声造影剂微泡的非线性效应的相关机理、动力学建模及其在超声医学领域中的应用研究也得到了极大的推动. 本文对下列课题进行了总结和讨论, 包括: 1)基于Mie散射技术和流式细胞仪对造影剂微泡参数进行定征的一体化解决方案; 2)通过对微泡包膜的黏弹特性进行非线性修正, 构建新的包膜微泡动力学模型; 3)探索造影剂惯性空化阈值与其包膜参数之间的相关性; 以及4)研究超声联合造影剂微泡促进基因/药物转染效率并有效降低其生物毒性的相关机理.     

New insights on the role of ROS in the mechanisms of sonoporation-mediated gene delivery

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity.     

Optical and acoustic monitoring of bubble cloud dynamics at a tissue-fluid interface in ultrasound tissue erosion

Short, high-intensity ultrasound pulses have the ability to achieve localized, clearly demarcated erosion in soft tissue at a tissue-fluid interface. The primary mechanism for ultrasound tissue erosion is believed to be acoustic cavitation. To monitor the cavitating bubble cloud generated at a tissue-fluid interface, an optical attenuation method was used to record the intensity loss of transmitted light through bubbles. Optical attenuation was only detected when a bubble cloud was seen using high speed imaging. The light attenuation signals correlated well with a temporally changing acoustic backscatter which is an excellent indicator for tissue erosion. This correlation provides additional evidence that the cavitating bubble cloud is essential for ultrasound tissue erosion. The bubble cloud collapse cycle and bubble dissolution time were studied using the optical attenuation signals. The collapse cycle of the bubble cloud generated by a high intensity ultrasound pulse of 4-14 micros was approximately 40-300 micros depending on the acoustic parameters. The dissolution time of the residual bubbles was tens of ms long. This study of bubble dynamics may provide further insight into previous ultrasound tissue erosion results.     

Ultrasound-induced cell lysis and sonoporation enhanced by contrast agents.

The enhancement of ultrasound-induced cell destruction, lysis, and sonoporation in low cell concentration suspensions (2 x 10(5)/mL) by the presence of contrast agents (gas bubble to cell ratio = 230) was demonstrated using cervical cancer cells (HeLa S3) suspensions containing micron-size denatured albumin microspheres filled with air (Albunex) or octafluoropropane (Optison). The suspensions were insonificated by 2-MHz continuous or tone burst ultrasound in near field. The spatial peak-pressure amplitude was 0.2 MPa. The enhancement of cell destruction due to Optison was shown to be much higher than that due to Albunex for similar bubble concentration and ultrasound conditions. For tone burst exposures, significant lysis and sonoporation only occurred in the presence of a contrast agent. The majority of the bioeffects observed occurred in the first 5 min of exposure. The relationship between the enhancement of bioeffects and duty cycle of tone burst ultrasound appears to indicate that both stable gas spheres of contrast agents and cavitation nuclei created by the disruption of the gas spheres play a significant role in causing the bioeffects.     

The influence of porosity on ultrasound attenuation in carbon fiber reinforced plastic composites using the laser-ultrasound spectroscopy

Wideband acoustic spectroscopy with a laser ultrasound source for quantitative analysis of the effect of porosity on the attenuation coefficient of longitudinal acoustic waves in carbon fiber reinforced plastic (CFRP) composite materials was experimentally implemented. The samples under study had different bulk-porosity levels (up to 10%), which were determined using X-ray computer tomography. A resonance ultrasound attenuation peak associated with the one-dimensional periodicity of the layered composite structure was observed for all samples. The absolute value of the resonance-peak maximum and its width depend on the local concentration of microscopic isolated pores and extended delaminations in the sample structure. The obtained empirical relationships between these parameters of the frequency dependence of the ultrasound attenuation coefficient and the type of inhomogeneities and their volume concentration can be used for rapid evaluation of the structural quality of CFRP composites.