这项工作得到了威康信托基金赠款088419 MDB和赞助施乐辉英国有限公司

1。涂层表面与基体的配体<ol><li>将超声波应用于细胞在3.5厘米的水井，无论是作为个人的菜肴，或作为一个6孔板。生化检测，直接将塑料外套配。免疫，熬盖玻片三次MilliQ水和4盖玻片放入每口井的底部。 </li><li>必须是衍生的玻璃表面，以确保有效的配体涂层。磺-M-maleimidobenzoyl-N-hydrosuccinimide在PBS酯溶解在1毫米。加入2毫升，每口井和30分钟，在室温下孵育。 </li><li>用PBS洗衍生表面的3倍。 </li><li>大衣或者衍生的玻璃或未经处理的塑料与整合配。每口井，适用于2毫升10微克/毫升整合ligand9，溶解在含Ca 2 +，Mg 2 +的 PBS过夜孵育在4°C。 </li><li>加热1％BSA的PBS溶解，85℃，13分钟准备monoparticulate BSA的。允许冷却，然后通过0.45微米的过滤器。 </li><li>配体涂层表面洗净与30分钟monoparticulate的BSA的PBS和块的3倍。 </li></ol> 2。单元的制备<ol><li>要控制整个实验的基质环境，应阻止蛋白质的合成增加25微克/毫升放线菌酮80％融合成纤维细胞为2小时。 </li><li>放线菌酮处理的细胞，用PBS冲洗脱离文化瓶使用0.5 mg / ml的胰蛋白酶/ EDTA。 </li><li> 500XĞ离心5分钟，离体细胞收获。 </li><li>重悬细胞在DMEM/25毫米肝素钠，25微克/毫升放线菌酮。 </li><li>暂停使用血球计数细胞密度，并稀释至105毫升，1。 </li><li>在37°C孵育20分钟的时间来恢复表面整合的细胞。 </li><li>冲洗配体涂层，牛血清白蛋白封闭的井用PBS 3倍。 </li><li>种子2毫升细胞悬液，将每口井。 </li><li>异体瓦特细胞散布在2小时37℃，5％的CO 2。 </li></ol> 3。超声波刺激<ol><li>温暖超声波发射器阵列和耦合凝胶至37°C。 </li><li>应用到每个发射器的耦合凝胶豌豆大小的BLOB。 </li><li>测试使用超声指标二极管探测器，每个发射器的功能。 </li><li>将发射阵列上井。返回数组到37°C，5％的CO 2，允许温度平衡10分钟。 </li><li>对超声信号的切换。 </li><li>超声波信号将关闭20分钟后，模仿治疗制度。如果需要刺激时间较短，板应该从数组中删除在适当的时间点。在所有情况下使用无刺激板块作为阴性对照。 </li></ol> 4。免疫荧光粘着分析<ol><li>粘着形成的分析，适用于20分钟的超声波信号然后让细胞保持在37℃，5％CO 2的结构，以发展为40分钟。 </li><li>吸媒体和修复细胞，用2毫升4％多聚甲醛的PBS。在室温下孵育14分钟。 </li><li>吸聚甲醛，并用PBS冲洗3次，申请下来的PBS以及边缘轻轻地避免产生剪切力。 </li><li>每个盖玻片转移以及从3.5厘米到24孔板为随后的免疫荧光染色。 </li><li>淬火残留多聚甲醛PBS 0.5毫升0.1 M甘氨酸应用24口井，每年。在室温20分钟孵育。 </li><li>吸出甘氨酸，用PBS冲洗三次。 </li><li> Permeabilise用0.5毫升0.5％的Triton X-100（W​​ / V）在PBS的细胞。在室温下孵育4分钟。 </li><li>分泌物的Triton X-100，用PBS冲洗三次。 </li><li>用3毫升0.5％（W / V），在PBS BSA的封锁。 4＆D孵育过夜例如，C。 </li><li>染色纽HVIN-1 BSA的块1:400稀释抗体含有粘着。在室温下孵育一小时。 </li><li>用PBS冲洗3次。 </li><li>应用荧光标记的二抗（1:200稀释）和TRITC标记的鬼笔环肽（1:200稀释）在BSA块肌动蛋白染色，（DyLight 488）。在室温30分钟孵育。 </li><li> PBS和一次水冲洗三次。 </li><li>上玻片安装在一个倒置的使用延长金奖mountant的位置。 </li></ol> 5。量化Rac1的激活下拉含量<ol><li> Rac1的活化分析，适用于20分钟的超声波信号到细胞在37℃，5％CO 2。评估的响应时间超过20分钟时，申请了20分钟的超声波信号，并保持细胞在37℃，5％CO 2剩余时间允许响应发展。 </li><li>抽吸媒体和地方以及S的冰。 2毫升冷PBS和吸冲洗井。确保任何残留的PBS离开1分钟后彻底抽吸倾斜的井被删除。 </li><li>裂解细胞在冰上35μL裂解液（10％甘油，20毫米HEPES pH值7.4，140 mM氯化钠，1％NP40，0.5％去氧胆酸钠，EGTA 4毫米，4毫米EDTA，1×完整的蛋白酶抑制剂），每口井。在这个例子中，6口井，每时间点生成细胞裂解液210μL。可以使用更多或更少的井，但总裂解液量应占〜200μL。 </li><li>刮细胞与细胞刮刀彻底，以确保完整的细胞裂解和离开井倾斜1分钟，允许裂解池。 </li><li>转移裂解物到一个冰冰冷的1.5毫升离心管（池同等待遇制度裂解）和自旋在21000×G 2分钟，在4°C颗粒细胞碎片。 </li><li> 180μL裂解液转移到冰冷的1.5 ml离心管含有130μL裂解液和25μLPAK-过剩athione琼脂糖珠10。随后凝胶分离，保持在-20°C的剩余原油裂解。 </li><li>捕捉PAK-谷胱甘肽琼脂糖珠的积极Rac1的旋转管时间为40分钟，在4°C。 </li><li>收获琼脂糖珠偶联于2000年通过离心1分钟×4Ğ°C。 </li><li>洗珠偶联用500μl裂解液三次，每次冲洗后收获2000X g和丢弃上清液离心珠。小心地取出200μL移液器任何剩余的溶解洗涤缓冲。 </li><li>洗脱每个样品35μL的SDS-PAGE上样缓冲液孵育85°C间为5分钟，加入上晃动热块。在21000×g下离心1分钟，取出的珠子。 </li><li>通过SDS-PAGE解决珠洗脱液，转移至硝酸纤维素和Rac1的探头。 </li></ol> 6。代表结果在这个协议中，我们描述了感应纽染色编辑粘着超声和Rac1活动。粘着实验的基线位置是成纤维细胞上配体α5β1 -整合传播没有形成，除非第二个降解syndecan-4，纤维连接蛋白受体，纽含粘连（ 图3A），此外，从事可溶性配体（ 图3B）3,4。然而，用超声波刺激诱 ​​导粘着形成的syndecan-4（ 图3C）参与相同的程度，表明，超声波刺激可以代替某些部件的纤维连接蛋白依赖的信号通路5。这个协议的主要障碍消除粘着在兴奋剂的情况下形成的。由放线菌酮抑制蛋白质合成的不完整，将允许自动刺激细胞是足够的基质沉积。细胞过度拥挤，也可导致低级别的粘着formation和细胞 - 细胞和细胞外基质的接触之间的串扰超声反应不佳将复杂的实验，旨在孤立单一的刺激。作为一个发展的基本实验中，我们表现出同样的实验，反复降解syndecan-4（ 图4）成纤维细胞与缺乏。这些成纤维细胞仍然响应超声（ 图4C），但未能作出回应降解syndecan-4配体（ 图4B）。使用Sdc4 - / -成纤维细胞的实验表明，超声波的确可以绕过某些纤维连接蛋白受体的需要，并演示了如何简单的协议，可以开发，以获得有关的信号通路的额外信息。 我们的生化实验表明，超声波可诱导Rac1的激活，使用下拉检测，德尔波索等 10所描述的方法是适应。 Rac1的活性0，10，30和60分钟后，INI降水超声刺激tiation显示，Rac1的活性峰在10-30分钟，然后再返回到基线（ 图5）。纽原油裂解印迹确保时间点之间的等效负载。结果类似于Rac1的激活参与降解syndecan-4 3，但略多于长期激活纤维连接蛋白。因此，通常需要8-10个重复，以实现重要数据。 Rac1的激活是细胞粘着形成的承诺负责，新生粘着开始形成10-30分钟，正好与Rac1的激活。一旦启动，这些粘连会继续成熟，变成大斑块粘附在图3和图4所示。 <img alt="图1" src="/files/ftp_upload/4024/4024fig1.jpg" /> 图1。超声波的形式。超声包括间歇性的1.5 MHz信号，由于低振幅（30毫瓦/厘米&lt;SUP&gt; 2，空间平均时间平均），有没有供暖效果。 <img alt="图2" src="/files/ftp_upload/4024/4024fig2.jpg" /> 图2。用超声波细胞的制备和刺激的工作流程示意图表示。 <img alt="图3" src="/files/ftp_upload/4024/4024fig3.jpg" /> 图3。超声诱导成纤维细胞粘着形成。成纤维细胞上的整合纤维连接蛋白结合片段刺激纤维连接蛋白降解syndecan-4-结合片段（B）或超声（三）前（一）传播。 60分钟的刺激之后，细胞固定，纽蛋白和肌动蛋白染色，萤光成像。酒吧= 10微米。 <img alt="图4" src="/files/ftp_upload/4024/4024fig4.jpg" /> 图4。缺乏降解syndecan-4的成纤维细胞粘着形成的超声诱导。 <em&gt; Sdc4 - / -成纤维细胞上的整合纤维连接蛋白结合片段刺激纤维连接蛋白降解syndecan-4-结合片段（B）或超声（三）前（一）传播。 60分钟的刺激之后，细胞固定，纽蛋白和肌动蛋白染色，萤光成像。虽然Sdc4 - / -成纤维细胞纤维连接蛋白不敏感，仍然超声诱导粘着形成，表明超声绕过基质受体参与。酒吧= 10微米。 <img alt="图5" src="/files/ftp_upload/4024/4024fig5.jpg" /> 图5。超声Rac1的激活。刺激细胞6口井，每时间点为0，10，30和60分钟。 GTP-Rac1的样品目前的金额从Rac1的下拉检测时间当然是可视化的孵化与反Rac1蛋白抗体上的印迹（顶端图像）。带的强度（平均8-10复制）量化揭示增加Rac1的1ctivity产生超声波信号，在10和30分钟，在60分钟（图），然后返回到基线水平。误差棒代表SEM

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安德鲁·哈里森是的施乐辉英国有限公司的雇员。

在这个协议中，我们描述了可以在基于细胞的实验中使用的方法，通常应用于人类患者的治疗。最终的目标是可以提炼治疗的分子机制，使了解超声波行动的。在这个协议中，我们使用小鼠胚胎成纤维细胞（MEFs）作为细胞模型系统，但超声也已被发现在人包皮成纤维细胞的主要5，间质干细胞，成骨细胞和软骨细胞6有效。我们用一个例子生化检测Rac1的激活，但同样的方法可以用来测试调节蛋白磷酸化或通过免疫蛋白复合物的形成。为免疫的例子，我们证明上粘着形成的影响超声，但可以审查任何分子的再分配。例如，可以测试的细胞膜，细胞质因素招聘蛋白酶colocalisation插件贩运囊泡或超声检查对有丝分裂纺锤体组织的影响。到目前为止，我们只限于自己来测试纤维连接蛋白依赖性途径，通过超声波对细胞的影响，胶原蛋白，或生长因子的存在，可以测试超声在复杂的环境影响细胞的行为如何建立一个图片类似于在体内的情况。一个更大的挑战将是检验超声使用时间推移成像，在场发射超声额外的技术障碍阻止光路和振动的影响。然而，治疗中的应用刺激，每天只需要20分钟的事实表明，一阵刺激后的细胞行为的分析，很可能是富有成效的。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>磺化-M-maleimidobenzoyl N-hydrosuccinimide的酯</td><td> FisherPerbio </td><td> PN22312 </td><td>溶解在水中的25毫米的股票，可以储存在-20°C </td></tr><tr><td> 6孔培养板</td><td>康宁</td><td> 3516 </td><td>从其他公司的塑料外套不佳</td></tr><tr><td> 13毫米的玻璃盖玻片</td><td>科学实验室用品Ltd.SLS </td><td> MIC3336 </td><td>从其他公司的玻璃外衣不佳</td></tr><tr><td> PBS </td><td>西格玛</td><td> D8537 </td><td></td></tr><tr><td> PBS， 钙+镁+ </td><td>西格玛</td><td> D8662 </td><td></td></tr><tr><td> 50K整合素配体（纤维连接蛋白片段） </td><td>构造描述和预paration在9，11 </td><td></td><td>替代矩阵的配体，可用于其他途径询问</td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3059 </td><td></td></tr><tr><td>放线菌酮</td><td>西格玛</td><td> C7698 </td><td>可以存储为10mg/ml的股票在水</td></tr><tr><td> DMEM/25毫米肝素钠</td><td>西格玛</td><td> D6171 </td><td></td></tr><tr><td> exogen 4000 + </td><td>史密斯和侄子公司</td><td></td><td></td></tr><tr><td>超声耦合凝胶</td><td>史密斯和侄子公司</td><td></td><td></td></tr><tr><td> SAFHS指标</td><td>史密斯和侄子公司</td><td></td><td></td></tr><tr><td> HVIN-1 </td><td>西格玛</td><td> V9264 </td><td></td></tr><tr><td> DyLight 488标记的抗鼠</td><td>思达科技</td><td> 715-485-150 </td><td></td></tr><tr><td> TRITC-标记的鬼笔&lt;/ TD&gt; <td>西格玛</td><td> P1951 </td><td></td></tr><tr><td>延长黄金的抗淬灭剂</td><td> Invitrogen公司</td><td> P36930 </td><td></td></tr><tr><td>完整的蛋白酶抑制剂（EDTA的自由） </td><td>罗氏公司</td><td> 056 489 001 </td><td>储存在-20°C 100×股票，由平板</td></tr><tr><td> PAK-谷胱甘肽琼脂糖珠</td><td>描述和准备建设10 </td><td></td><td></td></tr><tr><td>甘油</td><td>费舍尔</td><td> G/0650/17 </td><td></td></tr><tr><td> HEPES </td><td>阿波罗科学</td><td> BI8181 </td><td> 1M股票和pH值至7.4 </td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> S/0160/65 </td><td></td></tr><tr><td> NP40 </td><td>西格玛</td><td> I3021 </td><td></td></tr><tr><td>去氧胆酸钠</td><td>西格玛</td><td> D6750 </td><td></td></tr><tr><td> EGTA </td><td>西格玛</td><td> E4378 </td><tD&gt; </td></tr><tr><td> EDTA的</td><td>西格玛</td><td> E5134 </td><td></td></tr></tbody></table>

induction of adhesion-dependent signals using low-intensity ultrasound

在多细胞生物体中，细胞的行为是通过与细胞外基质的相互作用决定的。矩阵参与范围从调节细胞迁移和增殖，分泌和即使分化的后果。基本每个这些复杂的过程所产生的信号从细胞外基质受体的细胞表面分子间的相互作用。整合的原型受体与提供细胞外基质和细胞骨架之间的机械连接，以及发起的一些粘附依赖的信号转导通路。然而，它正变得越来越明显，额外的跨膜受体旁边的整合功能，调节下游的整合和信号。这些例子中最优雅的跨膜蛋白多糖，降解syndecan-4，α5β1整合与合作过程中粘附到纤维连接蛋白在体内模型demonstr吃的syndecan-4信号的重要性，作为降解syndecan-4基因敲除小鼠表现出愈合迟缓，由于低效的成纤维细胞迁移1,2。在野生型动物，所引发的纤维连接蛋白的外观，从受损的毛细血管渗漏和沉积在损伤组织巨噬细胞迁移的成纤维细胞向伤口。因此有极大的兴趣，增强纤维连接蛋白依赖的信号，并可以加快修复过程中发现战略。 纤维连接蛋白依赖的信号介导的整合和降解syndecan-4-介导的组件可以通过刺激细胞分离与重组纤维连接蛋白片段。虽然整合的参与是必不可少的细胞粘附，某些纤维连接蛋白依赖的信号受降解syndecan-4。降解syndecan-4激活Rac1的前伸的信号，使整合再分配1，触发细胞骨架分子，如纽，招聘，联络粘连，从而诱导定向迁移3。我们寻找替代战略激活这些信号，并发现，低强度脉冲超声波（LIPUS）可以模仿的syndecan-4参与的影响。在这个协议中，我们描述了30毫瓦/平方厘米 ，1.5兆赫超声波，1 kHz时（ 图1）脉冲，可应用于文化中的成纤维细胞（ 图2）诱导Rac1的激活和粘着形成的方法。超声刺激，最多为20分钟，这个参数的组合已被发现是最有效的加速临床骨折修复6。该方法采用重组纤维连接蛋白片段进行α5β1整合，没有参与的syndecan-4，需要由放线菌酮抑制蛋白质的合成，阻止成纤维细胞额外的基质沉积。，Ø积极作用f在修复机制的超声是有据可查的7,8，了解文化的超声分子的影响，我们应该能够改进治疗技术，以提高临床疗效。

Watch this Scientific Journal Video about Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound at JoVE.com

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

本协议描述成纤维细胞与低强度脉冲超声波的刺激，从而推动粘着形成和Rac1激活通过模仿跨膜基质受体，降解syndecan-4参与。这种方法允许在细胞水平上一个成功的临床技术的调查，从而提供了完善的治疗机会​​。

诱导粘附依赖使用低强度超声波信号

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

induction-of-adhesion-dependent-signals-using-low-intensity-ultrasound

Research

JoVE Journal

Bioengineering

9.4K 次观看.  University of Bristol. 本协议描述成纤维细胞与低强度脉冲超声波的刺激，从而推动粘着形成和Rac1激活通过模仿跨膜基质受体，降解syndecan-4参与。这种方法允许在细胞水平上一个成功的临床技术的调查，从而提供了完善的治疗机会​​。

视频: Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

超声造影有针对性的治疗胶质瘤的小鼠通过药物轴承纳米粒子交付和微血管消融

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

科研

生物工程

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

有针对性的超声微泡销毁技术简介

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高强度的实时监控聚焦超声（HIFU）消融<em&gt;体外</em&gt;使用谐运动成像的聚焦超声犬肝脏（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

通过光纤使用波前整形多个信号的传输

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

超声弹性成像验证腹主动脉水凝胶组织模拟幻影的研制

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

功能颅内多普勒超声监测脑血流

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

使用超声成像的对比增强次谐波辅助压力估计 （SHAPE），重点是识别门静脉高压症

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

用于表征用于超声触发药物释放的荧光标记微气泡的多时间尺度显微镜方法

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...