The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

对于与体积手持光声探测器操作的详细过程描述如下。此过程是根据关于动物和人体试验批准机构法规进行。 1.系统准备<ol><li>为〜15分钟的预热期之前的操作进行切换的激光，用于稳定输出光束。 </li><li>将水包围部分与隔离膜，其是在与皮肤（ 图1）接触。 注：在隔离膜（与皮肤接触），并与换能器（球形探针的中心）的最大灵敏度的区域之间的距离确定了有效的成像深度。 </li><li>填充的绝缘膜，并与去离子水的换能器的表面之间的约100毫升的，由一泵装置的整体体积。 </li><li>确保没有漏水，也没有气泡prese新台币。可替换地，通过提供的水再循环避免气泡。 </li><li>执行该实验在室温和确保耦合介质（水）被维持在该温度下。 </li></ol> 2.影像学准备<ol><li>人体成像的准备。 </li><li>从要被成像的部分具有脱毛洗剂，以避免在图像中的不期望的背景去除毛发（此步骤是可选的）。 </li><li>应用超声凝胶上的区域周围的皮肤，以提供高效的声耦合成像。把光声探测器中的感兴趣区域。确保没有气泡存在于所述超声耦合凝胶。 </li><li>动物成像的准备。 </li><li>确保护理和实验步骤与动物都在与机构和政府规章制度的协议。 </li><li>取下动物的皮毛在该区域的成像与剃须乳液。保护动物的眼睛兽医软膏，以防止干燥，并损害暴露于强脉冲激光辐射。 </li><li>用氯胺酮/甲苯​​噻嗪（100毫克/公斤公斤氯胺酮+ 5mg / kg的甲苯噻嗪KG）在实验前或使用异氟醚麻醉（2-3％（按体积计）的腹膜内注射（IP）的使用0.9升麻醉动物/在实验过程中最小气体流）。通过检查动​​物的后肢的反射确认麻醉。 </li><li>应用超声凝胶上的区域周围的皮肤进行成像，以提供高效的声学耦合，并把光声探测器中的感兴趣区域。确保没有气泡存在于所述超声耦合凝胶。 </li></ol> 3.预视图操作模式<ol><li>建立690纳米和900纳米，10至50赫兹的脉冲重复率之间的成像波长（多个）。选择该参数用于声音数据采集系统 - 1MΩ输入阻抗。获得2030样本对于每个激光脉冲以每秒和12位垂直分辨率40兆次采样的采样率。触发采集与激光Q开关输出。 </li><li>确保操作者和患者使用适于光激发波长（多个）护目镜。设置激光功率，使得光注量在组织表面保持在低于20毫焦/厘米2的实验对近红外波长中，以满足人体实验19安全暴露限制，并防止热应变和皮肤损伤的动物。 </li><li>开始预视图软件具有GPU执行的处理算法，以允许可视化三维图像在对应于激光的脉冲重复速率的帧速率。 </li><li>移动探针和/或以优化的可视化性能和定位感兴趣的结构将被成像的对象。 </li></ol> 4.数据采集<ol><li>数据采集​​扫描（手持）模式。 </li><li>如果需要的话，前采集注射的造影剂来丰富在感兴趣的区域中的对比度。 注：在我们的实验中，我们并没有进行对比度增强人体成像。然而，不同的造影剂可被潜在地用于此目的。吲哚菁绿（ICG）是一种可以在对成人2毫克/千克体重的最大推荐剂量被用于对比增强临床上认可的光造影剂的一个实例。 </li><li>启动数据采集与维护预览软件的执行在3.1中描述的参数的硬件。轻轻移动的成像区域周围的探针来跟踪感兴趣的结构。 注意：当同时采集在多个波长的激光的图像，在手持模式探测运动的速度，必须显著降低（优选低于2毫米/秒为，为了避免在光谱上未混合的图像中的运动有关的伪影的激光脉冲重复为50Hz速率）。 </li><li>数据采集​​的固定模式。 </li><li>所述成像对象（ 例如 ，动物）和手持式探头安装到所述保持器，并开始获取与保持在预视图中软件的执行在3.1中描述的参数。 </li><li>保持的光声探测器，并在实验过程中相同的位置的可视化中的感兴趣区域的动态生物事件的摄像部。 </li><li>注入造影剂，以跟踪其在感兴趣的区域动态分配。 注意：在我们的小鼠实验中，吲哚菁绿（ICG）用于对比度增强。作为一般准则，10纳摩尔或0.4毫克的量/公斤的ICG必须被引入到小鼠循环，以创建具有在体内多光谱optoacoustics可检测的对比度。 注：对比剂必须是批准用于由各个授权人和/或动物使用。 </li></ol> 5.最后确定实验<ol><li>停止激光。 </li><li>从成像区域去除的光声探测器。对动物的研究中，停止麻醉供应。 </li><li>定位红外加热器下的动物保持温暖，并防止与其他动物的接触，直到其从麻醉中完全恢复。从麻醉恢复过程中不要将动物无人值守。 </li></ol> 6.离线数据处理<ol><li>加载包含用于数据处理的软件应用程序所获取的光声信号中的文件（多个）。 </li><li>使用重构算法，以获得对应于所述光吸收对各帧和各波长的体积图像的三维矩阵​​阵列。 注意：对于重建它是优选使用的算法占扭曲因素，如heterogene成象物体在伊蒂埃斯和衰减，最终带宽和检测元件和光注量的变化的几何形状，为了效果，得到的吸收能量的分布更加定量表示。 </li><li>使用一个去混合算法来获得，从每个多波长帧，一组新的三维矩阵​​阵列表示的光吸收为存在于样品中的每个吸收物质。 </li><li>如果需要的话，进一步的处理表示光吸收分布，以便可视化的生物相关的参数和读取的矩阵阵列。 </li></ol>

<ol>
	<li>Stritzker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus-mediated+melanin+production+allows+MR+and+optoacoustic+deep+tissue+imaging+and+laser-induced+thermotherapy+of+cancer.">Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (9), 3316-3320 (2013).</li><li>Herzog, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Imaging+of+Cancer+Heterogeneity+with+Multispectral+Optoacoustic+Tomography.">Optical Imaging of Cancer Heterogeneity with Multispectral Optoacoustic Tomography.</a> Radiology. 263 (2), 461-468 (2012).</li><li>Johnson, S. P., Laufer, J. G., Zhang, E. Z., Beard, P. C., Pedley, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Differential+Tumour+Vascular+Pathophysiology+in+Vivo+by+Photoacoustic+Imaging.">Determination of Differential Tumour Vascular Pathophysiology in Vivo by Photoacoustic Imaging.</a> Eur J Cancer. 48, 186-187 (2012).</li><li>Yao, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+photoacoustic+computed+tomography+of+mouse+brain+metabolism+in+vivo.">Noninvasive photoacoustic computed tomography of mouse brain metabolism in vivo.</a> Neuroimage. 64, 257-266 (2013).</li><li>Strohm, E. M., Berndl, E. S. L., Kolios, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+label-free+photoacoustic+microscopy+of+single+cells.">High frequency label-free photoacoustic microscopy of single cells.</a> Photoacoustics. 1 (3-4), 49-53 (2013).</li><li>Beard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+photoacoustic+imaging.">Biomedical photoacoustic imaging.</a> Interface Focus. 1, 602-631 (2011).</li><li>Wang, L. H. V., Hu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+Tomography:+In+Vivo+Imaging+from+Organelles+to+Organs.">Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs.</a> Science. 335 (6075), 1458-1462 (2012).</li><li>Xiang, L. Z., Wang, B., Ji, L. J., Jiang, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-D+Photoacoustic+Tomography.">4-D Photoacoustic Tomography.</a> Sci Rep-Uk. 3, 10-1038 (2013).</li><li>Buehler, A., Dean-Ben, X. L., Claussen, J., Ntziachristos, V., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+optoacoustic+tomography+at+video+rate.">Three-dimensional optoacoustic tomography at video rate.</a> Optics express. 20 (20), 22712-22719 (2012).</li><li>Dean-Ben, X. L., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adding+fifth+dimension+to+optoacoustic+imaging:+volumetric+time-resolved+spectrally-enriched+tomography.">Adding fifth dimension to optoacoustic imaging: volumetric time-resolved spectrally-enriched tomography.</a> Light: Science and Applications. 3, (2014).</li><li>Fronheiser, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optoacoustic+monitoring+and+three-dimensional+mapping+of+a+human+arm+vasculature.">Real-time optoacoustic monitoring and three-dimensional mapping of a human arm vasculature.</a> J Biomed Opt. 15 (2), (2010).</li><li>Buehler, A., Kacprowicz, M., Taruttis, A., Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+handheld+multispectral+optoacoustic+imaging.">Real-time handheld multispectral optoacoustic imaging.</a> Opt Lett. 38 (9), 1404-1406 (2013).</li><li>Yang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+functional+photoacoustic+and+ultrasonic+endoscopy+of+internal+organs+in+vivo.">Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo.</a> Nat Med. 18 (8), 1297-1302 (2012).</li><li>Kruger, R. A., Lam, R. B., Reinecke, D. R., Del Rio, S. P., Doyle, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoacoustic+angiography+of+the+breast.">Photoacoustic angiography of the breast.</a> Med Phys. 37 (11), 6096-6100 (2010).</li><li>Heijblom, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+breast+cancer+using+the+Twente+photoacoustic+mammoscope:+What+do+we+learn+from+twelve+new+patient+measurements.">Visualizing breast cancer using the Twente photoacoustic mammoscope: What do we learn from twelve new patient measurements.</a> Optics express. 20 (11), 11582-11597 (2012).</li><li>Dean-Ben, X. L., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portable+spherical+array+probe+for+volumetric+real-time+optoacoustic+imaging+at+centimeter-scale+depths.">Portable spherical array probe for volumetric real-time optoacoustic imaging at centimeter-scale depths.</a> Optics express. 21 (23), 28062-28071 (2013).</li><li>Dean-Ben, X. L., Ozbek, A., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+real-time+tracking+of+peripheral+human+vasculature+with+GPU-accelerated+three-dimensional+optoacoustic+tomography.">Volumetric real-time tracking of peripheral human vasculature with GPU-accelerated three-dimensional optoacoustic tomography.</a> IEEE transactions on medical imaging. 32 (11), 2050-2055 (2013).</li><li>Dean-Ben, X. L., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+optoacoustic+human+angiography+with+handheld+video+rate+three+dimensional+scanner.">Functional optoacoustic human angiography with handheld video rate three dimensional scanner.</a> Photoacoustics. 1 (3-4), 68-73 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American National Standards for the Safe Use of Lasers ANSI Z136.1.. , (2000).</li><li>Ntziachristos, V., Razansky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+by+means+of+multispectral+optoacoustic+tomography+(MSOT).">Molecular imaging by means of multispectral optoacoustic tomography (MSOT).</a> Chemical reviews. 110 (5), 2783-2794 (2010).</li><li>Luke, G. P., Yeager, D., Emelianov, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+Applications+of+Photoacoustic+Imaging+with+Exogenous+Contrast+Agents.">Biomedical Applications of Photoacoustic Imaging with Exogenous Contrast Agents.</a> Ann Biomed Eng. 40 (2), 422-437 (2012).</li></ol>

The authors have nothing to disclose.

通过光声成像技术在小动物的研究提供了独特的优势创造了强大的动力转换的技术应用到临床实践中，有许多诊断和设想如治疗监测的应用。在乳腺癌和皮肤癌，炎症或外周血管疾病。然而，相对于小鼠或更小的动物，其可通过照射源和检测元件的足够数量的被包围，以创建一个有效的断层图像采集几何结构，对人体和限量光学穿透阻碍实现全身的大尺寸光声断层成像类似于MRI或CT检查。?...

光声（光声）成像每年吸引从生物和医学研究界日益增长的兴趣，这表现在数量不断增加的出版物涵盖各种各样的利用由技术1-5所提供的独特优点的新的应用程序。特别是，能力与在深度远远超出光的扩散极限高时空分辨率的图像进行频谱独特的光吸收剂打开前所未有的功能，用于功能和分子成像6-10。 事实上，光声技术应用到临床实践中译本自带的诊断和许多疾病的治疗监测前景十分广阔。然而，在光散射光子吸收的组织和与光声现象相关联的通常弱响应的有限传播限制了该方法的适用的深度。其结果是，手持optoacoustic探针已试图从本体11,12的外部接近的图像部分，而内窥镜系统所使用的通过自然孔口13插入它们提供从身体内的图像。一些低吸收人体的部位，如女性的乳房，并且也是由断层光声扫描仪14,15进行访问。特别令人感兴趣的是手持式的办法，因为它使大量的通用性，类似于超声。在这里，普通超声线阵探头，光声成像的改编依然充满挑战，主要是由于超声波和optoacoustics之间的断层成像要求根本性的分歧。而在标准超声高帧率被顺序使能发送接收计划采用在kHz范围内的高的脉冲重复频率，实时三维光声成像是通过同时采集的容积断层数据从单个int实现errogating激光脉冲。因此，高品质的光声成像意味着采集的三维数据从成象物体周围尽可能大的立体角。 最近，我们推出了第一台手持式光声探测三维（立体）影像实时16。该系统是基于二维的排列后的球面（蓝点在图1A中）256压电元件覆盖90°的角度阵列上。约为3×3 平方毫米的各个元件的尺寸，以及它们的取向和频率带宽（约2-6兆赫）从围绕球体的中心（黑立方体图一厘米刻度体积保证有效信号采集1A）。摄像区域的光激发设置有通过所述阵列的中央圆柱形腔插入的纤维束，以使任何波长SUSC通过对纤维束被传输的eptible可用于成像。换能器的阵列沿与光纤束的实际图像显示在图1B中 。有效激发并同时检测信号允许深层组织成像单次激励（1激光脉冲），从而在通过激光的脉冲重复频率确定的帧速率实时成像进一步与一个图形 - 启用处理单元（GPU）执行重建程序17。一个圆筒形壳体具有一个透明聚乙烯膜（ 图1C）连接到所述换能器阵列，以围成一个声学传输液体介质（水）。该膜进一步耦合到所述组织通过声凝胶的装置。的光声探测器作为手持操作模式下使用的一个图象被显示在图1D中 。 该证明THRee值维手持式光声成像结​​合实时功能成像能力来与对临床诊断和一些潜在应用的重要优点可以设想的各种迹象，如外周血管病，淋巴系统疾病，乳腺癌，皮肤损害，炎或关节炎18。此外，快速成像能力使与设置在一个固定的位置上的探头动态生物事件的可视化。结合快速波长调谐光学参量振荡器（OPO）激光技术，该方法允许光吸收剂的生物分布的实时成像。因此，新的可能性，可能会同样出现在小动物成像应用， 例如 ，在学习组织的血流动力学， 体内细胞跟踪，药代动力学，器官灌注可视化，有针对性的肿瘤和心血管系统，神经影像学或分子成像。

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

代表性的结果，显示了所描述的容积手持式光声探测器的功能，是展示在本节。在所有情况下，光积分通量在皮肤表面上保持在低于20毫焦/厘米2 19中的安全性的暴露极限。 探针在实时跟踪外围人脉管系统的性能被展示在图2中，在此实验的过程中，所述探针缓慢地沿着健康人类志愿者的手，在800纳米的激光器工作在单波长扫描以每秒17 10个脉冲，?...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

通用型手持式三维光声成像探测深部组织人力造影和功能的临床前研究中实时

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K 次观看.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

视频: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

手持式实时非侵入性小动物成像的临床光声成像系统

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...