作者感谢中国国家自然科学基金（61822505;11774101;61627827:81630046）、中国广东省科技规划项目（2015B020233016）和广州科技计划（201902001）的资助。

所有动物实验均按照中国广州华南师范大学动物护理与使用委员会提供的指南进行。
1. 系统设置
<ol><li>光学路径 （图 1）<ol>
<li>使用 532 nm 脉冲激光作为系统激光源。将激光的重复速率设置为 10 kHz，输出能量设置为 100%，使用用户定义的程序将触发器设置为外部触发器。</li>
		<li>通过光纤耦合器 （FC1） 将激光束耦合到单模光纤 （SMF）。在二维机动阶段（电机，最大速度:20 mm/s）上使用光纤校准器 （FC2） 对激光束进行协调。</li>
		<li>使用双轴气压计扫描仪（Galva）偏转激光束。使用可移动镜（M1）反射光束。通过 4×目标透镜聚焦光束（OL，数光圈:0.1）。</li>
		<li>使用XY翻译器安装（TM）修复自制的中空超声波传感器（UT，中央频率:25 MHz:带宽:90%以上:中心孔:3毫米）在OL12的底部。将聚焦光束穿过超声波传感器的中心孔。</li>
	</ol>
</li>
	<li>扫描路径<ol>
<li>在 WIM 期间使用现场可编程门阵列 （FPGA 2） 锁定 Galva。通过用户定义的程序设置适当的扫描范围和扫描速度。</li>
		<li>在 RIM 期间，使用现场可编程门阵列 （FPGA 1） 锁定电机。使用 FPGA 2 设置扫描频率和扫描点数。使用用户定义的程序来控制开始并停止扫描。</li>
	</ol>
</li>
	<li>数据采集<ol>
<li>使用 50 dB 放大器 （AMP） 放大 PA 信号。通过数据采集卡 （DAQ） 对信号进行数字化。通过 FPGA 1 或 FPGA 2 获取触发信号。</li>
		<li>使用图形处理单元 （GPU） 处理数据并行显示图像13。</li>
	</ol>
</li>
	<li>CCD 成像系统<ol>
<li>使用环形白色 LED（颜色温度:6500 K;光亮度: 40000 勒克斯:直径:7.5厘米）作为照明源。取出 M1，使用固定镜面 （M2） 反射光线。</li>
		<li>使用 PA 成像系统上的 CCD 摄像机（630 万像素）记录图像。用显示软件显示图像。</li>
	</ol>
</li>
</ol>2. 系统对齐
<ol><li>选择一个水箱（10厘米×10厘米×4.4厘米;下窗:3厘米×3厘米）。使用聚乙烯膜覆盖整个水箱（膜厚:10μm）。加入足够的超纯水。</li>
	<li>将水箱放在工作台上。</li>
	<li>打开激光开关。选择激光控制程序。预热5分钟。按泵开关上的"打开"按钮。将激光参数设置为每个步骤 1.1.1。打开激光的挡板。</li>
	<li>选择 A 线收集程序。按"开始"按钮捕获单点信号，并显示当前 A 线信号的振幅和频谱。</li>
	<li>将刀片放在水箱底部。将UT的底部浸入水箱中进行声学耦合。避免在UT的底部出现气泡。</li>
	<li>调整 Galva 的位置，在 UT 和 OL 之间调整 XY 翻译器以避免振荡信号，并确保这是共处的。</li>
	<li>调整工作阶段的高度，最大化信号的振幅，确定对焦位置。</li>
</ol>3. 动物实验
<ol><li>使用体重为 20+u201230 g 的 5+u20126 周大 BALB/c 鼠标。</li>
	<li>在实验前用注射的尿素（1克/千克）麻醉动物。</li>
	<li>在 WIM 和 RIM 之间进行过渡。<ol>
<li>使用平面超声波传感器。使用修剪器和脱毛霜剃掉鼠标背面的毛皮。将鼠标放在支架上（8 厘米× 2.8 厘米× 2 厘米）处于易发位置。</li>
		<li>使用超声波凝胶使成像区域与聚乙烯膜接触。避免接触部分出现气泡。</li>
		<li>将支架放在工作台上进行声学耦合。按照步骤 2.3\u20122.4 启动激光并收集 A 线信号。按照步骤 2.6\u20122.7 对齐。按"停止"以在对齐后结束集合。</li>
		<li>选择 WIM 程序。命名新创建的文件夹。将扫描参数设置为 20 mm/s 在"扫描速度"选项卡中，"扫描区域"选项卡中的"20 mm*20 mm"，以及"步骤"选项卡中的"20"。单击 "收集 "按钮开始扫描。</li>
		<li>单击 "停止" 按钮以在获取后结束扫描。单击 返回为零 ，将电机恢复为零。关闭激光挡板。将触发器设置设置为内部触发器。按下抽水开关的 关闭 按钮。</li>
		<li>将 WIM 扳机替换为 RIM 触发器，并将其连接到外部激光扳机。按下用于泵送开关的 开启 按钮。将触发器设置设置为外部触发器。单击 "退出 "按钮以退出 WIM 程序。</li>
		<li>使用步骤 2.4 收集 A 线信号。打开激光挡板。按照步骤 2.6\u20122.7 对齐。对齐后按 停止 以结束收集。</li>
		<li>选择 RIM 程序。命名新创建的文件夹。单击 "收集 "按钮开始扫描。</li>
		<li>单击 "停止" 按钮以在完成获取后结束扫描。单击 "退出 "按钮以退出 RIM 程序。</li>
		<li>成像结束时使用宫颈脱位对动物实施安乐死。</li>
	</ol></li>
	<li>进行血管可视化的 WIM。<ol>
<li>使用聚焦超声波传感器（中央频率:25 MHz:带宽:90%以上:焦距:8毫米）。去除老鼠耳朵或头皮的头发。<ol>
<li>使用手术刀在鼠标颅骨时间顶部的横向侧面（深入到头骨）上做一个小切口。使用眼科剪刀从此切口开始。切开头骨外侧的头皮。压缩出血点以止血。用普通盐水清洗伤口。将鼠标放在支架上。</li>
		</ol>
</li>
		<li>使用超声波凝胶使成像区域与聚乙烯膜接触。避免接触区域的气泡（补充图 1）。</li>
		<li>将支架放在工作台上进行声学耦合。使用步骤 2.3=u20122.4 打开激光并收集 A 线信号。使用步骤 2.6\u20122.7 对齐。对齐后按 停止 以结束收集。</li>
		<li>选择WIM程序。命名新创建的文件夹。在"扫描速度"选项卡中将扫描参数设置为"10 mm/s"，在"扫描区域"选项卡下将扫描参数设置为"10 mm*10 毫米"，在"步骤"选项卡中将扫描参数设置为"10 mm/s"。单击 "收集 "按钮开始扫描。</li>
		<li>单击 "停止" 按钮以在完成获取后结束扫描。单击 返回为零 ，使电机返回为零。单击 "退出 "按钮以退出 WIM 程序。</li>
		<li>在动物仍在麻醉期间，在手术结束时对动物实施安乐死。</li>
	</ol></li>
	<li>进行RIM对小动物进行动态监测。<ol>
<li>剃老鼠腹部的头发。将鼠标放在支架上处于超平位置。</li>
		<li>使用超声波凝胶使成像区域与聚乙烯膜接触。避免接触区域中的气泡。</li>
		<li>将支架放在工作台上进行声学耦合。执行步骤 2.3=u20122.4 启动激光并收集 A 线信号。执行步骤 2.6=u20122.7 以对齐。对齐后按 停止 以结束收集。</li>
		<li>选择 RIM 程序。命名新创建的文件夹。单击 "收集 "按钮开始扫描。</li>
		<li>单击 "停止" 按钮以在完成获取后结束扫描。单击 "退出 "按钮以退出 RIM 程序。</li>
	</ol></li>
	<li>使用 RIM 数据，通过用户定义的程序沿深度方向重建最大振幅投影 （MAP）。观察动物的动态变化。</li>
</ol>

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所有动物实验均按照机构动物护理和使用委员会批准的准则和条例进行。作者在手稿中没有相关的经济利益，也没有其他潜在的利益冲突可以披露。

在这里，我们提出了一个双栅格扫描光声小动物成像仪非侵入性血管可视化，这是设计和开发，以捕捉血管结构和相关的血液动态变化。DRS-PAI 的优点是将 WIM 和 RIM 集成到一个系统中，从而更容易研究小动物的血管动态和血管网络结构。该系统可提供高分辨率广域血管可视化和实时血液动力学。
在目前的系统中，光激发采用单波长光源实现。未来的多波长系统将提供其他参?...

在基础生物医学领域，小动物可以模拟人类的生理功能。因此，小动物成像在指导人类同源性疾病研究、寻求有效治疗等方面起着重要作用。光声成像 （PAI） 是一种非侵入性成像技术，结合了光学成像和超声成像2的优点。光声显微镜（PAM）是小动物3的基础研究的宝贵成像方法。PAM可以根据光学激发和超声波检测4轻松获得高分辨率、深渗透、高特异性和高对比度图像。
具有特定波长的脉冲激光被组织内源性色谱吸收。随后，组织温度升高，导致产生光诱发的超声波。超声波可以通过超声波传感器检测。经过信号采集和图像重建，吸收器的空间分布可达到5.一方面，全器官血管网络的可视化需要广阔的视野。广域扫描过程通常需要很长时间才能确保高分辨率6，7，8。另一方面，观察小动物的血动力学活动需要快速的实时成像。实时成像有利于实时研究小动物的生命体征9、10、11。实时成像的视野通常足够小，以确保较高的更新率。因此，在实现广阔的视野和实时成像之间往往存在权衡。以前，两个不同的系统分别用于广域成像或实时成像。
这项工作报告了双栅格扫描光声成像仪 （DRS-PAI），它集成了基于二维机动转换阶段的广域成像和基于双轴电表扫描仪的实时成像。进行宽场成像模式 （WIM） 以显示血管形态。对于实时成像模式 （RIM），目前有两个功能。首先，RIM 可以提供实时 B 扫描图像。通过测量沿深度方向的血管位移，可以揭示呼吸或脉搏的特征。其次，RIM 可以定量测量 WIM 图像中的特定区域。通过提供当地 WIM 区域的可比图像，可以准确显示当地变化的细节。该系统在血管可视化的广域成像和局部动态的实时成像之间设计了灵活的过渡。该系统在基础生物医学研究中是可取的，因为那里需要小动物成像。

<table>
	<tbody>
		<tr>
			<td>12 bit multi-purpose digitizer</td>
			<td>Spectrum</td>
			<td>M3i.3221</td>
			<td>Data acquisition card</td>
		</tr>
		<tr>
			<td>A-line collected program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>RF Bay</td>
			<td>LNA-650</td>
			<td>Amplifier</td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Veet</td>
			<td>33-II</td>
			<td>Animal depilatory</td>
		</tr>
		<tr>
			<td>Fiberport Coupler</td>
			<td>Thorlab</td>
			<td>PAF-X-7-A</td>
			<td>Fiber Coupler</td>
		</tr>
		<tr>
			<td>Field Programmable Gate Array</td>
			<td>Altera</td>
			<td>Cyclone IV</td>
			<td>Trigger Control</td>
		</tr>
		<tr>
			<td>Fixed Focus Collomation Packages</td>
			<td>Thorlabs</td>
			<td>F240FC-532</td>
			<td>Fiber Collimator</td>
		</tr>
		<tr>
			<td>Foused ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics Processing Unit</td>
			<td>NVIDIA</td>
			<td>GeForce GTX 1060</td>
			<td>Processing data</td>
		</tr>
		<tr>
			<td>Holder</td>
			<td>Self-made</td>
			<td></td>
			<td>Animal fixation</td>
		</tr>
		<tr>
			<td>Laser control program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Guangdong Medical Laboratory Animal Center</td>
			<td>BALB/c</td>
			<td>Animal Model</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Mshot</td>
			<td>MS60</td>
			<td>CCD camera</td>
		</tr>
		<tr>
			<td>Microscope Objective</td>
			<td>Daheng Optics</td>
			<td>GCO-2111</td>
			<td>Objective Lens</td>
		</tr>
		<tr>
			<td>Mirror</td>
			<td>Daheng Optics</td>
			<td>GCC-1011</td>
			<td>Moveable/Fixed Mirror</td>
		</tr>
		<tr>
			<td>Moving Magnet Capacitive Detector Galvanometer Scanner</td>
			<td>Century Sunny</td>
			<td>S8107</td>
			<td>real-time scanner</td>
		</tr>
		<tr>
			<td>Mshot image analysis system</td>
			<td>Mshot</td>
			<td></td>
			<td>Display software</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>CR DOUBLE-CRANE</td>
			<td>H34023609</td>
			<td>Normal Saline</td>
		</tr>
		<tr>
			<td>Ophthalmic Scissors</td>
			<td>SUJIE</td>
			<td></td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Planar ultrasonic transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Wrap</td>
			<td>HJSJLSL</td>
			<td></td>
			<td>Polyethylene Membrane</td>
		</tr>
		<tr>
			<td>Program Control Software</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined Program</td>
		</tr>
		<tr>
			<td>Pulsed Q-swithched Laser</td>
			<td>Laser-export</td>
			<td>DTL-314QT</td>
			<td>532-nm pulse Laser</td>
		</tr>
		<tr>
			<td>Real-time imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>Ring-shaped white LED</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Codos</td>
			<td>CP-9200</td>
			<td>Animal Shaver</td>
		</tr>
		<tr>
			<td>Single-Mode Fibers</td>
			<td>Nufern</td>
			<td>460-HP</td>
			<td>Single-mode fiber</td>
		</tr>
		<tr>
			<td>Surgical Blade</td>
			<td>SUJIE</td>
			<td>11</td>
			<td>Blade</td>
		</tr>
		<tr>
			<td>Surgical Scalpel</td>
			<td>SUJIE</td>
			<td>7</td>
			<td>Scalp Remove</td>
		</tr>
		<tr>
			<td>Translation Stage</td>
			<td>Jiancheng Optics</td>
			<td>LS2-25T</td>
			<td>wide-field scanning stage</td>
		</tr>
		<tr>
			<td>Ultrasonic Transducer</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>GUANGGONG PAI</td>
			<td>ZC4252418</td>
			<td>Acoustic Coupling</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Tokyo Chemical Industry</td>
			<td>C0028</td>
			<td>Animal Anestheized</td>
		</tr>
		<tr>
			<td>Water tank</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field imaging program</td>
			<td>National Instrument</td>
			<td>LabVIEW</td>
			<td>User-defined program</td>
		</tr>
		<tr>
			<td>XY Translator Mount</td>
			<td>Self-made</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dual raster-scanning photoacoustic small-animal imager for vascular visualization

小动物血管网络成像在基础生物医学研究中发挥了重要作用。光声成像技术在小动物影像学中具有巨大的应用潜力。小型动物的广域光声成像可以提供具有高空间分辨率、深度渗透和多重对比度的图像。此外，实时光声成像系统可观测小动物血管的血动力学活动，可用于研究小动物生理特征的动态监测。在这里，展示了一个双栅格扫描光声成像仪，具有可切换的双模成像功能。广域成像由二维机动转换阶段驱动，而实时成像则由电表实现。通过设置不同的参数和成像模式，可以进行小动物血管网络的体内可视化。实时成像可用于观察药物引起的脉搏变化和血流量变化等。广域成像可用于跟踪肿瘤血管的生长变化。这些在基础生物医学研究的各个领域都很容易被采用。

DRS-PAI 的示意图显示在 图 1中。该系统允许WIM与RIM之间灵活且可重复的切换。收购的 PA 信号处理得很快，以生成 PA B 扫描和 MAP 图像。CCD 相机可以提供样品的照片。
DRS-PAI 的所有组件均集成并组装在成像仪设置中（图 2），便于组装和操作。在 WIM 中，使用二维机动阶段的连续栅格扫描。记录运行阶段的信号。数据采集是在统一翻?...

Watch this Scientific Journal Video about Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization at JoVE.com

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

设计了双栅格扫描光声成像仪，集成了广域成像和实时成像。

用于血管可视化的双栅格扫描光声小动物成像仪

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

dual-raster-scanning-photoacoustic-small-animal-imager-for-vascular

Research

JoVE Journal

Bioengineering

4.0K 次观看.  South China Normal University. 设计了双栅格扫描光声成像仪，集成了广域成像和实时成像。

视频: Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

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

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

使用光声血流循环黑色素瘤细胞的检测和隔离

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging technologies. Existing retinal imaging modalities, such as fundus photography1, confocal scanning laser ophthalmoscopy (cSLO)2, and optical coherence tomography (OCT)3, have significant contributions in monitoring disease onsets and progressions, and developing new therapeutic strategies. However, they predominantly rely on the back-reflected photons from the retina. As a consequence, the optical absorption properties of the retina, which are usually strongly associated with retinal pathophysiology status, are inaccessible by the traditional imaging technologies.
Photoacoustic ophthalmoscopy (PAOM) is an emerging retinal imaging modality that permits the detection of the optical absorption contrasts in the eye with a high sensitivity4-7 . In PAOM nanosecond laser pulses are delivered through the pupil and scanned across the posterior eye to induce photoacoustic (PA) signals, which are detected by an unfocused ultrasonic transducer attached to the eyelid. Because of the strong optical absorption of hemoglobin and melanin, PAOM is capable of non-invasively imaging the retinal and choroidal vasculatures, and the retinal pigment epithelium (RPE) melanin at high contrasts 6,7. More importantly, based on the well-developed spectroscopic photoacoustic imaging5,8 , PAOM has the potential to map the hemoglobin oxygen saturation in retinal vessels, which can be critical in studying the physiology and pathology of several blinding diseases 9 such as diabetic retinopathy and neovascular age-related macular degeneration.
Moreover, being the only existing optical-absorption-based ophthalmic imaging modality, PAOM can be integrated with well-established clinical ophthalmic imaging techniques to achieve more comprehensive anatomic and functional evaluations of the eye based on multiple optical contrasts6,10 . In this work, we integrate PAOM and spectral-domain OCT (SD-OCT) for simultaneously in vivo retinal imaging of rat, where both optical absorption and scattering properties of the retina are revealed. The system configuration, system alignment and imaging acquisition are presented.

Photoacoustic ophthalmology (PAOM), an optical-absorption-based imaging modality, provides the complementary evaluation of the retina to the currently available ophthalmic imaging technologies. We report the using of PAOM integrated with spectral-domain optical coherence tomography (SD-OCT) for simultaneous multimodal retinal imaging in rats.

集成光声眼底镜检查和谱域光学相干断层扫描

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Photoacoustic Cystography

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The risk of bladder cancers in children due to radiation exposure is more significant than many other cancers. Here we demonstrate the feasibility of nonionizing and noninvasive photoacoustic (PA) imaging of urinary bladders, referred to as photoacoustic cystography (PAC), using near-infrared (NIR) optical absorbents (i.e. methylene blue, plasmonic gold nanostructures, or single walled carbon nanotubes) as an optical-turbid tracer. We have successfully imaged a rat bladder filled with the optical absorbing agents using a dark-field confocal PAC system. After transurethral injection of the contrast agents, the rat's bladders were photoacoustically visualized by achieving significant PA signal enhancement. The accumulation was validated by spectroscopic PA imaging. Further, by using only a laser pulse energy of less than 1 mJ/cm2 (1/20 of the safety limit), our current imaging system could map the methylene-blue-filled-rat-bladder at the depth of beyond 1 cm in biological tissues in vivo. Both in vivo and ex vivo PA imaging results validate that the contrast agents were naturally excreted via urination. Thus, there is no concern regarding long-term toxic agent accumulation, which will facilitate clinical translation.

Photoacoustic cystography (PAC) has a great potential to map urinary bladders, a radiation sensitive internal organ in pediatric patients, without using any ionizing radiation or toxic contrast agent. Here we demonstrate the use of PAC for mapping urinary bladders with an injection of optical-opaque tracers in rats in vivo.

光声膀胱造影

Conventional pediatric cystography, which is based on diagnostic X-ray using a radio-opaque dye, suffers from the use of harmful ionizing radiation. The ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the optical mean free path (~1 mm in skin) with high resolution. By combining optical absorption contrast with the high spatial resolution of ultrasound in a single modality, this technique can penetrate deep tissues. Photoacoustic microscopy systems can have either a low acoustic resolution and probe deeply or a high optical resolution and probe shallowly. It is challenging to achieve high spatial resolution and large depth penetration with a single system. This work presents an AR-OR-PAM system capable of both high-resolution imaging at shallow depths and low-resolution deep-tissue imaging of the same sample in vivo. A lateral resolution of 4 &#181;m with 1.4 mm imaging depth using optical focusing and a lateral resolution of 45 &#181;m with 7.8 mm imaging depth using acoustic focusing were successfully demonstrated using the combined system. Here,&#160;in vivo&#160;small-animal blood vasculature imaging is performed to demonstrate its biological imaging capability.

Switchable Acoustic and Optical Resolution Photoacoustic Microscopy for In Vivo Small-animal Blood Vasculature Imaging

Here a switchable acoustic resolution (AR) and optical resolution (OR) photoacoustic microscopy (AR-OR-PAM) system capable of both high resolution imaging at shallow depth and low resolution deep tissue imaging on the same sample in vivo is demonstrated.

可切换声光学分辨率光声显微镜<em&gt;体内</em&gt;小动物血液血管成像

Photoacoustic microscopy (PAM) is a fast-growing invivo imaging modality that combines both optics and ultrasound, providing penetration beyond the ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

高性能紧凑型光声层析成像系统<em&gt;体内</em&gt;小动物脑成像

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

用于血管可视化的双栅格扫描光声小动物成像仪

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