作者想感谢斯文持有人及康利先生在这项研究中的宝贵援助。这项工作是由部分由美国国立健康的授权号码R00CA134920]美国肺脏协会的授权号码RG-194681-N]。 NinePoint医药公司赞助出版成本与这篇稿子。

1。成像系统对外直接投资的技术细节已被描述以前4-6。周对外直接投资是在25和100之间的每秒帧数和每圆形的横截面图像在512和2048之间的轴向深度公司的成像速度进行。自定义2.4神父（直径为0.8 mm）本研究中所用的螺旋扫描导管被设计操作通过标准气管镜的进入口的。导管包括用于将光聚焦到支气管壁和一个一次性使用的外护套的内光核心。导管主体在成像过程中保持静止，而内芯是在25和100赫兹和翻译在拉回介于1.25和5毫米/秒的速度之间的速率旋转。在组织的系统的时间是6毫米的轴向分辨率，并提供了一种图像范围的深度为7.3毫米的4-6。导管为基础的对外直接投资在这项研究中，复制在体内支气管镜对外直接投资（FIgure 1）。然而，这个协议也可以被施加到成像与一个台式光学系统（ 图3和4）。 2。成像系统设置<ol><li>打开成像系统</li><li>设置和记录摄像参数（​​旋转速度，拉回速度，图像采集速率，等）。对于对外直接投资在这项研究中使用的成像系统，在10-50 fps的图像，获得。 </li><li>将导管旋转交界处，回调设备。 </li><li>旋转导管和检查图像质量。调整系统的调整和偏移根据需要。 </li></ol> 3。组织制备<ol><li>将一个桌面上的台式和一组肺标本垫一次性吸水垫。 </li><li>如果成像手术前病人体内样本，请务必咨询病理科，以确保所有切缘（支气管，血管和实质的利润）进行评估，记录，和/或删除的病理logist。 </li><li>确定切除标本在肺门支气管气道进入。删除任何明显的气道黏液内使用注射器孔。如果有必要，将一个长的塑料管段的注射器吸孔气道中的深。 </li><li>触诊的检体的外表面，以确定病变景点。 </li><li>使用细金属探测器，小心翼翼地浏览支气管树，直到附近的病变的利益。 </li><li>打开气道沿探头，直至病变的利益是看到或摸到下呼吸道粘膜。 </li><li>小心地取出任何气道黏膜的血液或粘液，用棉签覆盖病变。 </li><li>将对外直接投资导管上方的气道粘膜并获得的图像，以确认病变相关的气道粘膜并识别感兴趣的组织学相关的高品质的摄像区域。 </li></ol> 4。组织标记<ol> &lt;李的在步骤3.8以前的影像学表现的基础上气道的兴趣选择区域。 </li><li>选择两个点上的组织，沿着所希望的行的成像。点可能是平行于要么的长度（ 图2）或周向（ 图3）方面的气道，这取决于所需的结果。空间点相距不超过1.5厘米，使组织的部分可能适合在一个组织学块进行处理。如果一个组织长度&gt; 1.5厘米，然后分割成多个长1.5厘米签署地区的利益创建多个匹配的影像学检查：组织学对组织长度。 </li><li>浸细笔尖开放的孔针（ 即 25号7/8“长）标记染料（的三角生物医学科学，达勒姆，北卡罗莱纳州）的组织。 </li><li>小心地擦去多余的墨水用纱​​布关闭针的外部，仅在针孔离开组织标记油墨。 </li><li>在气道黏膜组织垂直穿刺选择点沿线的成像。 </li><li>重复步骤3.3至3.5的第二个点上呼吸道粘膜。 </li><li>如果墨水运行在距穿刺部位的粘膜表面，使用棉签小心地将多余的油墨。 </li><li>取出粘液或血液的表面上的气道粘膜用棉头，如果存在的话。 </li><li>如果圆周放置的墨点内的气道，它是有用的针开放双方扁平化的气道组织中的摄像视场（ 图3a）。 </li></ol> 5。影像组织<ol><li>将在每个墨水标记和图像，以确保标记是可见对外直接投资的对外直接投资导管。商标应显示为焦距与上覆高度散射粒子和快速的信号的衰减，这对应于在穿刺部位（ 图3b，图4a，图4g的墨粒子的基本结构的组织内的中断</stronG&gt;）。 </li><li>如果墨水标记（s）是不可见的对外直接投资，重复步骤4.3至4.7为不可见的标记。如果墨痕可见的对外直接投资，继续执行步骤5.3。 </li><li>平行的导管放置使得导管光学标记（ 图2b）超出第一墨水覆盖在组织上的气道粘膜表面的两个油墨标记。与一个轻量级的对象的导管的近端锚定和固定的前端可以帮助减少运动伪影。 </li><li>继续收集的对外直接投资回落。 </li><li>对外直接投资的回落图像，以确保两个墨痕中是可见的影像，以检查运动伪影（ 图3和图4）。如果标记是不可见的，重复步骤5.1至5.4。 </li></ol> 6。收集和处理组织<ol><li>将一个绿色的墨点（的三角生物医学科学，达勒姆，NC）的气道粘膜组织定位成像扫描开始的，0.3厘米距离日E Ink的标志，首先出现在成像回调（ 图2c）。 </li><li>删除组织涵盖了两个黑色的墨痕和绿色墨水标记。修剪组织，以符合标准的组织学处理盒。如果切割新鲜组织是困难的，那么组织可以固定前组织学检查，以消除组织。 </li><li>将组织在组织学处理盒和固定在10％福尔马林浸泡至少48小时。 </li><li>过程组织在一个组织的处理器，可通过任何组织学部门。 </li><li>嵌入石蜡组织中，使得切口部将是平行的两个气道表面上的黑色墨痕。 </li><li>使用组织切片面对的石蜡块，直到墨水标记是可见的，或整个组织的部分是可见的，以先到者为准。 </li><li>一旦这两个黑色墨水标记是可见的，切成5微米厚的部分，并安装到载玻片上。 </li><li>继续削减和安装5微米厚的部分50微米，直到黑色的墨痕不再是可见的或组织结束，以先到者为准。 </li><li>按照标准的苏木精伊红（H＆E）染色染色的协议和盖玻片幻灯片。 </li></ol> 7。影像处理如果图像获得与台式扫描仪或其他扫描技术，在一个单一的横截面的图像是可见的两个墨痕，然后将图像可以直接与相应的组织学相关。如果被收购的螺旋扫描导管立体数据集，图像将需要重新插值，使一个单一的2D图像一分为二的两个墨痕与组织学的相关性。这可以通过使用ImageJ或其他图像处理软件。油墨在某些情况下，可能无法轻易地可见的，在这种情况下，相邻的分段/幻灯片应检查。

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早期肺部恶性肿瘤的评估可以非常具有挑战性，由于缺乏症状，无法可视化早期肿瘤影像学或支气管镜。对外直接投资提供了组织学的分辨率，大面积组织微结构的三维视图实时2-6附近。对外直接投资支气管内已被证明在作为一种安全的技术，可用于获得高分辨率的体积数据集，在很长的气道段在肺呼吸道11-13（ 动画 ）患者。但是，只有很小的活检获得病理同行体内设置，没有提供足够的相关性肺部病理成像标准的发展对外直接投资的。为了准确地评估肺部成像中看到的对外直接投资的特点，它是必不可少的组织学的相关性，以获得精确匹配的图像。我们提出了一个简单而有效的方法精确，以邻对外直接投资与组织学网元之间的相关性气道成像的体外肺切除的标本，这是适用于几乎所有的体外组织类型。一旦成像标准已经建立了体外与匹配-酮组织学，这些标准可以然后被施加到体内成像。 组织染料用于标记的感兴趣区域成像清晰可见，在对外直接投资和组织学。通过使用简单的技术方向的组织，墨痕，可能会允许对外直接投资的功能和组织学的研究结果比较，以确定可识别的成像特点，组织病理成像和组织学相关。该技术是廉价和实用的，从而使它在许多光学成像应用有用。 在体内设置，如激光标记的方法，可以使用组织取向25。然而，T他体积小的支气管活检仍然是在体内研究制定具体的成像标准的肺部病理的一个限制因素。虽然体外研究可以作为适当的替代品在体内成像，有一定的局限性。 离体肺标本不充气往往显示外科手术引起的肺不张，改变了正常的肺泡结构的外观。在技​​术上具有挑战性，因为大多数外科手术肺组织病理冰冻切片后收到的评价在此期间，胸膜表面被破坏，干扰标本通货膨胀的充气手术切除的肺组织与组织，组织学相关的标记。非病理肺不张是没有看到在体内设置一个工件，从而此限制将不相关的体内肺成像。此外，由于缺乏可以使血管内的血液在体外标本难以distingu从其他信号无效结构十岁上下的血管结构。在体内设置，多普勒OCT / 26日至28日对外直接投资的结构OCT /对外直接投资的增加将有助于识别船只。 运动伪影可以看出，他们是不存在的体外体内 。这可能是潜在的问题，在标准的OCT系统的采集速率较慢。然而，对外直接投资的系统是目前快速的帧速率&gt; 200帧29-31。因此，预计不会运动伪影，将是一个重大的问题。前在体内成像OCT和对外直接投资研究的成功证明了精细成像功能的可视化14,15,18,19。 在这项研究中，我们已经证明了体积对外直接投资的相互关系，以评估肺部病理组织为基础的诊断。描述的步骤，目的是提供精确匹配的组织学，用作金STANDA路的对外直接投资的图像解释。 一旦特定的成像条件，经肺病理已开发和验证，与匹配的一对1组织学的体外的准则，然后可以被应用到后续的体内成像研究与使用支气管活检作为金标准评估的摄像功能可见一斑。肺切除标本的应用程序，但这种技术可以应用到几乎所有的组织类型，以提供精确的成像需要确定正常和病理组织的精细影像学特征组织学的​​相关性。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>组织标记染料</td><td>三角生物医学</td><td> TMD，TMD-BK-G </td><td></td></tr></tbody></table>

optical frequency domain imaging of ex vivo pulmonary resection specimens: obtaining one to one image to histopathology correlation

肺癌是癌症相关死亡的首要原因1。鳞癌和小细胞癌通常发生在与传导性气道，而腺癌通常更多的外围设备中的位置。肺恶性肿瘤发现，早在疾病过程中可能会很困难，因为有几个缺陷：放射性分辨率，支气管镜在评估潜在的气道黏膜组织，并确定早期病理变化的限制，和小样本的大小和/或不完整的组织学活检采样。高分辨率成像方式，如光频域成像（OFDI），提供非破坏性的，大面积组织显微结构的3维视图的深度接近2毫米的实时（ 图1）2-6。对外直接投资中使用的各种应用，包括评估冠状动脉粥样硬化6,7和食管肠上皮化生和不典型增生<sup6,8-10。 支气管镜OCT /对外直接投资已被证明是一个安全的工具，用于评估肺的气道11-23（ 动画 ） 在体内成像。 OCT已在肺部气道16,23 17,22实质的动物模型，并在人体内气道14,15评估。 OCT成像的正常气道已经证明气道的层次和肺泡附件的可视化和不典型增生病变的评价，已发现有用的区分等级的发育不良，支气管粘膜11,12,20,21。对外直接投资的成像支气管粘膜已被证明在很短的支气管段（0.8厘米）18。此外，体积对外直接投资跨越多个气道代在猪和人的肺在体内的气道已19。支气管OCT /对外直接投资通常采用薄，弹性的导管，这是与标准bronchos兼容科皮克接入端口。另外，OCT和对外直接投资基于针状探头最近已被开发，其可以用于图像区域以外的气道壁的肺或胸膜表面17的 。 没有研究，虽然OCT /对外直接投资已使用并证明是可行的， 在体内肺成像，精确匹配的一对对外直接投资：组织学已经完成。因此，具体的各种肺部病变的成像的标准还没有被开发。 在体内组织病理学同行只支气管镜活检小片段，这是难以与大对外直接投资的数据集。此外，他们还没有登记需要大容量的对外直接投资提供全面的组织学。因此，具体的影像学特征的肺部病理不能在体内设置。一对精确匹配，对外直接投资和组织学的相关性准确评估是非常重要的功能，看到在O外商直接投资对组织学为金标准，以获得特定的图像解释标准为肺部肿瘤和其他肺部病变。一旦具体的成像标准已经制定，验证体外一对一的一个组织学，标准可能在体内成像研究。在这里，我们提出了一个精确的，一对一之间的相关性在离体肺切除标本的高分辨率光学成像和组织学方法。在本手稿中，我们描述了所使用的技术，以配合对外直接投资的组织学图像。然而，这种方法是不特定的对外直接投资，任何光学成像技术可用于获得组织学类型的注册图像。我们进行了气道为中心，用专门的定制支气管镜2.4法国的对外直接投资（直径0.8mm）的导管。组织样品组织染料标记，对外直接投资与组织学可见。仔细取向程序被用来精确地关联伊马晋和组织取样的位置。在这个手稿的技术被用来进行体积对外直接投资的第一个示范的相互关系，以组织为基础的诊断评估肺部病理24。这种直接，有效的方法也可以推广到其他类型的组织提供精确的成像需要，以确定正常和病变组织的精细影像学特征组织学的​​相关性。

黑色的墨痕应该是在1 - 1.5厘米之间分开，以指示感兴趣的摄像区域。绿色油墨标记应放置在成像扫描开始之前，第一黑色墨水标记定位的检体（ 图2和图3a）。组织墨痕应该是可见的都对外直接投资成像和组织学（ 图3和4）。 （ 图3）在正常猪和人的气道（ 图4），典型的气道分层应该可见。作为薄的上皮细胞（E）是可见的，中度致密，均匀的层，在管腔方面的气道的信号。固有层由有组织的信号强烈信号不良的组织，如信号激烈的结缔组织包括弹性蛋白和胶原致发光（EL）的固有层（LP）的各种部件，对应信号不良的唾液型腺组织（G ）。信号有偶尔可见的差管（D）穿越呼吸ratory与支气管管腔上皮细胞连接。平滑肌出现不连续的，中间穿插平滑肌分册和是无法识别对外直接投资。的ħ＆Ê和三色渍，气道压条可以可视化（ 图3C，3D，3F，3克，4B，4C，4E，及4楼 ），在那里上三色的表面的致密的弹性和胶原组织出现深蓝色和的底层平滑肌肉污渍红色（SM）。软骨环（C）出现信号不佳的月牙状结构具有明确的界限，在猪呼吸道重叠和不重叠在人类呼吸道。周围的软骨环的软骨膜出现一层薄薄的信号激烈的组织，涵盖了信号不良的软骨环。在人类呼吸道的圆周（ 图4g和4h），肺泡附件（A）是显示为薄，信号激烈格子状肺泡壁与信号无效肺泡腔。固有层内的血管位是V，可见的信号无效的直线或圆弧结构底层的阴影轻度伪影（箭头）。 <img alt="图1" src="/files/ftp_upload/3855/3855fig1.jpg" /> 图1。猪呼吸道对外直接投资。 在体内获得的图像从猪气道机械通气。 （一）ODFI近端气道的横截面。 （二）对外直接投资远端气道的横截面。 （三）ODFI纵向节近端气道，高放大倍率的图像面板e以红色突出显示的区域。 （四）对外直接投资的纵向部分的远端气道，在更高的放大倍率图像的面板e绿色突出显示的区域。 （五）ODFI纵截面的气道从近端到远端（左到右）。导管的直径为0.8毫米的刻度代表0.5毫米为单位递增。虽然气道壁的不同层和肺泡附件是可辨别的对外直接投资图像，它是难以精确地解释的解剖合作rrelate的对外直接投资的信号没有直接登记注册的组织学。 E：上皮，LP：SM：固有层，黏膜下层，C：软骨，肺泡附件。 <img alt="图2" src="/files/ftp_upload/3855/3855fig2.jpg" /> 图2。 （一） 组织标记的猪呼吸道。打开气道腔面有两个黑色的墨痕，平行放置的纵向方面的气道，1.5厘米。 （二）对外直接投资的导管放在两个黑色墨水标记包括两个标志在对外直接投资的回调。 （三）气道与额外的绿色油墨标记定位在试样上的摄像扫描的开始。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/3855/3855fig3highres.jpg" src="/files/ftp_upload/3855/3855fig3.jpg" /> 图3。猪呼吸道的对外直接投资和组织学表现出精确的对应有关使用组织标记。（一）开放气道腔面有两个黑色的墨痕，平行放置的圆周方面的气道。引脚用于进一步开放气道（箭头）。 （二）对外直接投资的猪呼吸道都墨痕（星号），（三）正是相关组织学与H＆E染色（星号：黑色墨迹，可见上呼吸道上皮细胞）及（d）相关的三色染色可见。比例尺：2毫米。 （五）对外直接投资（F）对应的组织学与H＆E染色及（g）相关的三色染色图像的放大视图。 E：呼吸道上皮细胞，EL：致密的胶原和弹性组织，SM：平滑肌，C：软骨环（组织学的神器已经导致人工分离的软骨环），G：涎腺组织，D：进入涎腺导管上皮细胞。比例尺：250微米。 <a href="http://www.jove.com/files/ftp_upload/3855/3855fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图4" fo:content-width="4.75in" fo:src="/files/ftp_upload/3855/3855fig4highres.jpg" src="/files/ftp_upload/3855/3855fig4.jpg" /> 图4。对外直接投资和组织学人的呼吸道表现出精确的相关组织标记。（一）对外直接投资的人近端气道都墨痕可见（星号）。 （二）正相关的组织学用黑墨水与H＆E染色，标志着明显的呼吸道上皮细胞（星号）及（c）相关的三色染色。比例尺：2毫米。 （四）对外直接投资的图像放大视图及（e）相应的组织学与H＆E（F）三色染色。比例尺为250μm。 E：呼吸道上皮细胞，LP：固有层，G：涎腺组织，C：软骨环，邮编：软骨膜。在人类气道，典型的分层是可见的。疏松结缔组织内，有穿插红染色平滑肌（SM，板C和F分册），不形成连续的频带，从而对外直接投资作为一个独立的层中是不可见的。 （七）对外直接投资的人远端气道及（h）正是相关的H＆E组织学与黑色的墨痕，可见上呼吸道上皮细胞（星号）。比例尺：2毫米。肺泡附件（A）是作为信号激烈格​​子状肺泡壁与信号无效肺泡腔可见。固有层内的血管空间也可见作为信号空隙结构基本轻微的阴影（箭头）。

Watch this Scientific Journal Video about Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation at JoVE.com

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

对图像的方法<em&gt;体外</em肺切除标本与光频域成像（OFDI）和获得精确的组织学的相关性，这是必不可少的肺部病理制定具体的对外直接投资的解释标准。这种方法适用于其他类型的组织和成像技术，获得精确的成像，精确的图像解释和评估的组织学相关性。成像用这种方法建立的标准将适用于在未来的图像评估<em在体内</em研究。

光频域成像<em&gt;体外</em肺切除标本：获得一对一的图像病理组织学的相关性

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

optical-frequency-domain-imaging-ex-vivo-pulmonary-resection

Research

JoVE Journal

Bioengineering

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

14.0K 次观看.  Harvard Medical School. 对图像的方法体外

视频: Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

高分辨率三维成像 EX - VIVO生物样品显微CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

科研

生物工程

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 ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

一分钟，小一瓦光热消融肿瘤使用Porphysomes，内在多功能纳米囊泡

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

基于数字图像相关的骨折检测中股骨皮质应变的估算方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...