作者想感谢新加坡教育部资助的1级研究补助金 (RG48/16: M4011617) 和新加坡教育部资助的2级研究补助金 (ARC2/15: M4020238) 的财政支持。作者想承认 Dr. Rhonnie 奥地利 Dienzo 对动物处理的帮助。

所有动物实验都是根据新加坡南洋理工大学动物保育和使用委员会批准的指导方针和规定进行的 (动物协议号-SBS/NIE-A0263). 
 1. 手持式实时临床 PA 和美国成像系统 <ol> <li> 手持式临床排系统的示意图 33 显示在 图 1a 中。它由一个光学参量振荡器 (参量) 激光器泵浦的频率加倍的纳秒脉冲钕: YAG 激光器, 分岔光纤束 ( 图 1b ), custom-made 3D 打印手持探针架 ( 图 1c ) 33 , 临床双模态和 PA 系统, 和一个临床上兼容的线性阵列 (见材料表). </li>
	<li> 通过单击桌面上的软件图标, 运行制造商在临床美国系统中提供的软件. </li>
	<li> 从触摸屏中选择 #39; 研究和 #39; 按钮在研究模式下操作美国系统。单击脚本列表中的美国和 PA 组合成像脚本, 然后单击 "运行" 按钮以进行组合模式下的图像处理. </li>
	<li> 使用激光触发或使用光电二极管将临床美国系统与激光同步. 
	注意: 将固定的同步从激光器连接到美国系统的同步中。确保提供正晶体管-晶体管逻辑 (TTL) 信号作为同步信号。光电二极管信号也可用于同步目的。美国系统同步在连接到光电二极管探测器使用光电二极管偏置模块。无论何时激光, 光电二极管都发出信号来触发和同步激光器和美国系统。每次执行此步骤. </li>
	<li> 打开激光器, 接通交流电源, 然后将激光控制器上的键向左打开。在确保重复率为10赫兹 (F10 将显示在显示屏上) 和 Q 开关延迟是低至170和 #181; s, 以确保低激光能量。若要设置延迟, 请按选择键, 直到看到延迟值并将其增加到 170. 
	注: 激光大约需要20分钟来预热. </li>
	<li> 打开计算机上的软件界面, 然后在 "转到" 菜单中输入波长 675 nm, 然后按 #39; 启动和 #39; 按钮以 675 nm 设置波长. 
	注: 激光可调谐从670到 2500 nm, 但是, 它是不稳定的在 670 nm. </li>
	<li> 按下快门按钮并使用开关将激光束对准光纤输入. </li>
	<li> 使用1英寸 (2.5 厘米) 直径的焦距 15 mm 的平凸透镜, 将激光束聚焦到纤维束上, 这样所有的光就会落在光纤输入端上. 
	注: 光纤有1600小纤维捆绑在一起。它是中间分岔的两个矩形输出端, 其中有800光纤。800纤维被包装成一个面积 0.1 x 4 厘米, 以配合的尺寸。每个光纤的核心直径是185和 #181; m, 数值孔径为 0.22. </li>
	<li> 对齐后关闭激光器 </li>
	<li> 从4探头持照角度不同 (0 和 #176; 5 和 #176;, 10 和 #176;, 和15和 #176;) 根据应用程序选择合适的探头支架 (成像深度、对象大小、对象形状和对象的位置). 
	注: 探头的设计和3D 打印在实验室。它有三插槽, 两个分岔光纤和中央一个为一。探头座的尺寸基于光纤的尺寸和光的大小。对高深度成像淋巴结所需的光照进行了蒙特卡罗模拟研究。信噪比在较低深度为15和 #176 的光照较高; 33 . </li>
	<li> 将分岔光纤放入3D 打印探头支架上, 光照角度为15和 #730;. </li>
	<li> 插入支架的中间槽. 
	注: 图 1d 显示了带有光纤的探头支架的照片。线性阵列有128阵列元素。中心频率为 8.5 MHz, 小数带宽为95%。它的长度是3.85 厘米。然而, 该系统只有64并行数据采集硬件, 需要两个激光脉冲来收集所有128元素的数据。因此, 该系统的有效帧率是激光器的脉冲重复率的一半, 它是每秒 34 5 帧. </li>
	<li> 通过松开侧面的螺钉并在调整精确距离后将其拧紧, 以将光纤端和纤维末端之间的距离调整到1厘米. 
	注意: 参数优化的 SLN 成像与模拟和幻影实验 33 。可以用探针架上的两个螺钉固定。这给出了改变光纤和光的距离的灵活性. </li>
	<li> 在激光器上切换, 确保在前面的位置获得矩形激光光斑. </li>
	<li> 关掉激光器。增加激光能量 (通过增加延迟) 到期望值为成像目的. 
	注: 请参阅手册中的最大延迟, 可以设置为钕: YAG 激光器。该系统用于 SLN 成像的预期延迟值设置为 210. </li>
</ol> 2。分辨率特性 <ol> <li> 将商业上可用的鸡肉组织板切割成 6 x 6 cm 2 板。用刀子把它切成0.5 厘米厚的薄片. </li>
	<li> 放置一个点对象, 像23克直径0.6 毫米的针, 在鸡胸肉组织的顶端. </li>
	<li> 打开激光. 
	警告: 在使用激光处理协议的其余部分时, 应佩戴安全护目镜。由于激光能量较弱, 在对准过程中发生了异常. </li>
	<li> 在不同深度上将针的 PA 图像叠加在一起, 将厚度为0.5 厘米的多个鸡胸肉组织切片, 由 3 cm 组成. 在鸡胸肉组织层之间应用美国凝胶, 以改善美国的耦合. </li>
	<li> 将波束形成的图像保存并存储为. 席子文件. </li>
	<li> 关闭激光. </li>
	<li> 使用图像处理软件 in-house 代码处理数据 17 . 
	注意: 为了确定轴向和横向的分辨率, 从归一化 PA 信号中找出点传播函数, 并将它们与高斯分布函数 17 相匹配。获得最大宽度的一半。要获得点扩展函数, 需要对点进行图像处理。然而, 有另一种方法获得点传播功能时, 成像点目标是困难的 (在我们的情况下, 对于一个非常微小的点目标的信号是相当小的, 因此我们使用一个稍大的目标)。如果目标是大的, 而不是直接得到点扩展函数, 你可以得到一个边缘扩展函数。然后利用边缘展开函数的第一阶导数, 可以得到点扩散函数。因此, 使用点目标来计算分辨率 22 并不是绝对必要的. </li>
</ol> 3。SLN 成像的动物准备 p 类 = "jove_con帐篷 ">> 注意: 上述的手持临床成像系统被证明为成像小动物 SLN。为实验, 6-到8星期老健康, 雌性老鼠 (局: 大, 220 和 #177; 30 g) 被购买了。雌性大鼠使用的原因是雄性大鼠乳癌发生率较低。然而, 雄性大鼠也可用于研究。此外, 在文献中, 雌性大鼠更广泛地用于 SLN 成像. 
<ol> <li> 鼠麻醉 <ol> <li> 成像前, 麻醉的麻醉液, 其中含有氯胺酮鸡尾酒 (100 毫克/毫升), 嗪 (20 毫克/毫升), 和注射生理盐水的比例 2:1: 1。增加0.2 毫升的鸡尾酒每100克的动物重量到一个无菌手术1毫升注射器针 (27G, #189; 英寸). </li>
		<li> 用手把老鼠的颈部 right-lower, 用酒精拭子将腹部的象限消毒。将麻醉液注入动物体内. </li>
		<li> 通过检查脚趾捏的反射来确保动物麻醉. </li>
	</ol> </li> <li> 对于 SLN 图像, 从感兴趣的区域中轻轻地移除头发, 并提供商业可用的脱毛膏。尽可能多地使用所需的覆盖区域。在 3-5 分钟的应用后, 用湿棉签除去奶油。为防止眼部干燥和意外激光损伤, 应用人工泪液软膏. </li>
	<li> 将蓝色 underpad 放在桌子上, 并将动物放在它的侧面。通过鼻锥 (0.75% 的异氟醚和氧气 (1.2 升/分)) 进行吸入麻醉, 在实验过程中维持麻醉下的动物。将脉冲血到老鼠的后爪上, 以监测整个实验中的心率和周围氧饱和度. 
	#160; 注意: 请确保动物是温暖的使用的热垫批准动物使用. </li>
</ol> 4。 在体内 大鼠的 SLN 成像 <ol> <li> 成像前, 使用注射器将0.5 到1毫升的美国凝胶涂抹在皮肤上, 并使用一个涂抹器将其传播得很好。在成像区放置一个0.5 厘米厚的鸡胸肉组织切片, 大小 6 cm x 6 厘米, 并在鸡组织上应用更多的美国凝胶, 以改善耦合. 
	注: #160; LNs 位于皮肤下 (2-3 毫米以内) 的大鼠。在人类中, LNs 的深度是1厘米. 因此, 鸡的组织被放置在大鼠的成像区, 模仿人类的成像场景。另外, 组织模仿幽灵可以用来代替鸡的组织. </li>
	<li> 在激光器上切换。将手持式探头托架放在鸡肉组织的顶部, 并将其扫描 (将持有者右向左移动) 在组合的美国和 PA 模式下. 
	注: 激光束的面积约为3厘米 2 , 计算结果为大约8兆焦耳/cm 2 , 小于美国国家标准学会 (ANSI) 安全限制 (20 兆焦耳/cm 2 ) 35.在临床美国系统中, 成像深度设置为2厘米。当激光器打开时, 应随时佩戴安全护目镜. </li>
	<li> 将成像深度设置为2厘米以进行 PA 成像. </li>
	<li> 获取感兴趣区域的控制图像, 在胸腔上方的前腿上方, 通过在造影剂注射前从侧面移动手持探头. 
	注意: 所有数据都以波束形成的数据类型保存, 以便进一步处理. </li>
	<li> 在动物的前爪中注入0.2 毫升的造影剂 (即: 、MB (10 毫克/毫升)), 并对其进行良好的按摩2分钟, 以促进造影剂运动到淋巴结通过淋巴管. </li>
	<li> 在5分钟后, 用手持探针沿鸡组织扫描, 在 PA 信号的帮助下定位 SLN. 
	注意: 所有帧都以波束形成的数据类型保存. </li>
	<li> 单击 "#39; 冻结和 #39; 从美国系统上的控件按钮, 然后单击和 #39; 从触摸屏监视器导出选定的框架和 #39; 按钮导出记录的数据. 
	注意: 数据可以存储在不同的格式, 即波束形成, 扫描转换, 通道和智商 </li>
	<li> 在动物的顶端加2层0.5 厘米厚的鸡组织切片, 并找到 SLN, 以展示深度成像的可行性达1.5 厘米. </li>
	<li> 成像后, 取出所有的鸡组织切片 </li>
	<li> 关闭激光. </li>
</ol> 5。SLN 的 PA 光谱 <ol> <li> 在大鼠上放置一个0.5 厘米厚的鸡胸肉组织切片. </li>
	<li> 使用该软件将激光器的波长设置为 670 nm. </li>
	<li> 在激光器上切换。用探针沿该区域的鸡胸肉组织扫描, 借助 PA 信号定位 SLN. </li>
	<li> 在识别 SLN 后保持探头稳定. </li>
	<li> 在提供的激光调谐软件中, 在激光软件中输入波长为 800 nm, 将速度设置为 10 nm/秒, 然后单击并 #39; 启动和 #39; 按钮. 
	注意: 这将改变波长从 670 nm 到 800 nm 的速度 10 nm/秒。波长范围的变化取决于所使用的造影剂的吸收光谱。MB 在 670 nm 附近有一个尖峰. </li>
	<li> 观察 PA 信号随波长变化的变化. </li>
	<li> 关闭激光. </li>
	<li> 卸下鸡肉组织切片. </li>
</ol> 6。实时针跟踪使用排 <ol> <li> 在动物身上放置一个0.5 厘米厚的鸡胸肉组织切片。将波长设置为 675 nm. </li>
	<li> 在激光器上切换。在 PA 信号的帮助下, 移动探头在屏幕上定位和识别 SLN. </li>
	<li> 实时针跟踪 <ol> <li> 注入23克针, 尺寸为 0.6 x 32 mm 2 , 平行于通过鸡组织进入动物, 到达 SLN, 同时引导它在 Real-time 的临床美国系统监视器. </li>
	</ol> </li> <li> 在实验后关闭激光。从动物身上取出鸡组织和脉冲血, 将动物移到工作台上。用棉抹布清洗大鼠的超声波凝胶. </li>
	<li> 将动物放在它的床上用品上, 并监视它直到它恢复知觉. </li>
	<li> 当动物的行为正常后, 将其返回笼子. </li>
</ol>

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作者在稿件中没有相关的财务利益, 也没有其他潜在利益冲突的披露。

目前癌症的筛查、诊断和治疗的费用非常高。有不同的成像方式, 正在用于癌症筛查和诊断。然而, 这些成像技术有很多限制, 包括笨重的机器大小, 侵入性诊断, 友好病人, 太昂贵, 要求电离辐射, 或使用放射性造影剂。因此, 需要有一个高效、成本效益高、real-time 的成像和引导系统。结合美国和 PA 成像是一种技术, 可用于有效的, 非侵入性的筛查, 诊断和分期的癌症。临床 PA 成像可以使更可行的 FDA...

对于癌症的检测和分期, 有不同的成像技术。一些广泛使用的成像模式是磁共振成像 (MRI), x 射线计算机断层扫描 (CT), x 射线, 超声 (美国), 正电子发射断层扫描 (PET), 荧光成像,等1,2,3,4. 但是, 现有的一些成像技术要么是侵入性的、有害的辐射, 要么是缓慢的、昂贵的、笨重的或对病人不友善的。因此, 有一个不断需要开发新的, 快速的, 和 cost-effective 成像技术的诊断和治疗5。
光声成像 (排) 是一种新兴的成像技术, 它结合了丰富的光学对比度和高超声分辨率在更深的成像深度5,6,7,8, 9。在排, 一个短的激光脉冲用于组织照射。光被组织吸收, 导致小的温度上升。由于热弹性膨胀, 压力波 (以声波的形式) 在组织内产生。产生的声波 (也称为光声 (PA) 波) 是获得了一个宽带超声换能器 (一) 外的组织边界。这些获得的 pa 信号可以用来重建 pa 图像, 揭示组织内部的结构和功能信息。排有广泛的应用, 包括: 血管成像, 前哨淋巴结成像, 脑血管成像, 肿瘤成像, 分子成像,等10,11,12, 13,14,15排有许多应用, 因为它的优点是: 更深的穿透深度, 良好的空间分辨率, 和高的软组织对比度。在排的对比可以是内生的血液, 黑色素,等。当内源对比度不够强时, 外源对比剂如有机染料、纳米粒子、量子点、等16、17、18、19、20,21可用于改善对比度。
尽管排便与其他成像技术相比有许多好处, 但临床翻译仍然是一个很大的挑战。主要的限制是使用的激光器笨重的性质, 大多数用于数据采集的 USTs 与临床美国系统不兼容, 而且没有提供可供使用的美国临床成像系统, 可以访问原始通道数据.直到最近, 具有访问原始数据的商用临床美国机器才成为可用的22。在这项工作中, 我们的目的是展示使用一个临床美国平台的手持式安装的可行性。我们的目的是通过在小动物模型中显示前哨淋巴结 (淋巴结) 的非侵入性成像来证明这一点。
侵袭性乳腺肿瘤是女性癌症死亡的主要原因之一。早期诊断和分期乳腺癌是决定治疗策略的关键, 对患者的预后起着重要作用。乳腺癌分期前哨淋巴结活检 (SLNB) 通常使用23,24。SLN 是主要的淋巴结, 可能发现癌细胞是最高的, 由于转移。SLNBs 涉及注射染料或放射性示踪剂, 其次是用小切口切开该区域, 然后在染料的情况下或在盖革计数器的帮助下以视觉方式定位 SLN, 以防放射性示踪剂。经过鉴定, 一些 SLN 被删除的病理学研究24,25。阳性 SLNB 表明, 肿瘤已转移到附近的淋巴结和其他器官可能。阴性 SLNB 表明, 转移的概率是微不足道的26。SLNB 有许多相关的并发症, 如手臂麻木, 淋巴水肿,等27为了消除 SLNB 相关的并发症, 需要一种非侵入性的成像技术。
对于小动物和人类的 SLN 映射, PA 成像在不同对比剂的帮助下得到了广泛的探索15,28,29,30,31,32. 但是, 目前所使用的系统不能用于临床情况, 正如前面所指出的那样。另一个需要解决的问题是 SLNB28中涉及的外科手术过程。为了减少患者的恢复时间和副作用, 需要对细针穿刺活检 (FNAB) 进行微创手术。在这项工作中, 一个临床美国系统被用来结合美国和 PA 成像是使用。为了便于临床安装, 设计了一种定制的用于住宅光纤和光的手持支架。亚甲蓝 (MB) 用于识别和测绘淋巴结. 此外, 为了消除与 SLNB 手术相关的并发症, 还演示了无创 real-time 针跟踪。

<table>
	<tbody>
		<tr>
			<td>Q-switched Nd:YAG laser</td>
			<td>Continuum</td>
			<td>Surelite</td>
			<td>Pump laser</td>
		</tr>
		<tr>
			<td>Optical parametric oscillator</td>
			<td>Continuum</td>
			<td></td>
			<td>OPO laser</td>
		</tr>
		<tr>
			<td>Clinical ultrasound imaging system</td>
			<td>Alpinion</td>
			<td>E-CUBE 12R</td>
			<td>Dual modal ultrasound and photoacoustic imaging system</td>
		</tr>
		<tr>
			<td>Linear array ultrasound transducer</td>
			<td>Alpinion</td>
			<td>L3-12</td>
			<td>128 element linear array transducer with centre frequency of 8.5 MHz, fractional bandwidth of 95%,</td>
		</tr>
		<tr>
			<td>Bifurcated optical fiber</td>
			<td>CeramOptec</td>
			<td>Custom made</td>
			<td>To couple the light from the laser to the handheld fiber holder</td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Thorlabs</td>
			<td>LB1869</td>
			<td>Focus light from the laser to the optical fiber</td>
		</tr>
		<tr>
			<td>Ultrasound gel</td>
			<td>Progress/parker acquasonic gel</td>
			<td>PA-GEL-CLEA-5000</td>
			<td>Acoustic coupling</td>
		</tr>
		<tr>
			<td>Image Processing software</td>
			<td>Mathworks</td>
			<td>Matlab</td>
			<td>Home made program using Matlab</td>
		</tr>
		<tr>
			<td>Anesthetic Machine</td>
			<td>medical plus pte ltd</td>
			<td>Non-Rebreathing Anaesthesia machine with oxygen concentrator.</td>
			<td>Supplies oxygen and isoflurane to animal</td>
		</tr>
		<tr>
			<td>Pulse Oxymeter portable</td>
			<td>Medtronic</td>
			<td>PM10N with veterinary sensor</td>
			<td>Monitors the pulse oxymetry of the animal</td>
		</tr>
		<tr>
			<td>Animal distributor</td>
			<td>In Vivos Pte Ltd, Singapore</td>
			<td></td>
			<td>Animal distributor that supplies small animals for research purpose.</td>
		</tr>
		<tr>
			<td>Breathing mask</td>
			<td>Custom made</td>
			<td></td>
			<td>Used along with animal holder to supply anesthesia mixture to the animal</td>
		</tr>
		<tr>
			<td>chicken breast tissue</td>
			<td>Pasar</td>
			<td></td>
			<td>Used to add depth to mimic human imaging scenario</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td>BD Precisionglide</td>
			<td>23G,1 and half inch</td>
			<td>Used for realtime needle guidance</td>
		</tr>
		<tr>
			<td>Holder for the fiber optic cable</td>
			<td>Custom made</td>
			<td></td>
			<td>To hold the input end of the bifurcated cable</td>
		</tr>
		<tr>
			<td>Handheld probe</td>
			<td>Custom made 3D printed</td>
			<td></td>
			<td>With two slots for the two output ends of the optical fiber and one slot for the ultrasound transducer</td>
		</tr>
		<tr>
			<td>Methylene blue (10 mg/mL)</td>
			<td>Sterop</td>
			<td></td>
			<td>Contrast agent for PA imaging</td>
		</tr>
		<tr>
			<td>Laser tuning software</td>
			<td>Surelite OPO PLUS</td>
			<td>SLOPO</td>
			<td>Software to tune the wavelength of OPO laser</td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>SP05/M</td>
			<td>To detect the laser pulse to trigger the ultrasound system</td>
		</tr>
		<tr>
			<td>Photodiode bias module</td>
			<td>Thorlabs</td>
			<td>PBM42</td>
			<td>To amplify the photodiode signal to tigger ultrasound signal</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Reckitt Benckiser</td>
			<td>Veet</td>
			<td>Used to remove hair from the imaging area</td>
		</tr>
		<tr>
			<td>Laser power meter</td>
			<td>Ophir</td>
			<td>Starlite, p/n: 7Z01565</td>
			<td>Used to measure the laser power</td>
		</tr>
	</tbody>
</table>

hand-held clinical photoacoustic imaging system for real-time non-invasive small animal imaging

光声成像在临床中的翻译是一项重大挑战。手持 real-time 临床光声成像系统是非常罕见的。在这里, 我们报告一个联合光声和临床超声成像系统集成了一个超声探头与轻分娩的小动物成像。我们通过在小动物身上显示前哨淋巴结成像以及微创 real-time 针引导来证明这一点。一个具有原始通道数据的临床超声平台允许将光声成像集成到手持 real-time 的临床光声成像系统中。亚甲蓝用于前哨淋巴结成像 675 nm 波长。此外, 使用成像系统显示了双模态超声和光声成像的针制导。高达1.5 厘米的深度成像演示了10赫兹激光器的光声成像帧速率为每秒5帧。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56649/56649fig1.jpg"> 
图 1: 系统描述。(a)具有双模态临床美国系统的排系统的示意图表示。参量-光学参量振荡器, 光纤束, 跳频光纤支架, USM-临床美国机器。光纤支架集成了两个输出光纤束。在实验过程中, 使用异氟醚和氧的麻醉机来保持动物的麻醉。(b)分岔光纤的照片。i/o 表示光纤的输入端...

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Hand-held Clinical Photoacoustic Imaging System 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

一个临床手持光声成像系统将证明 real-time 非侵入性小动物成像。

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

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Research

JoVE Journal

Bioengineering

11.4K 次观看.  Nanyang Technological University. 一个临床手持光声成像系统将证明 real-time 非侵入性小动物成像。

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

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

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.

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 ...

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 ...

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 ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

应用交互式、患者特异性模型对神经纤维束进行深部脑刺激治疗的靶向性

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...

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

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

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 ...