Name: a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy
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Description: Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

This project was supported by Grant Numbers HL96593 from NIH and D56HP20783 from HRSA/ HHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or HRSA / HHS.

注意:获取处理用于获得毛细管图像先前已公布的和使用静止数字照相机与对应图像采集和分析的计算机程序来完成11 21本实验利用静止图像进行分析，而不是视频，简化用于分析图像采集。以下描述了一种用于从图像中定量的毛细血管的新技术。 1.图像增强处理<ol><li>获得的数字图像与单色的数码相机。通过取已知长度的对象的图片，例如用相机尺子校准图像以已知尺寸的目的。此过程允许计算机程序，测量和加工后准确地计算毛细管。理想情况下最高的精度，应使用一个手提袋（科学制造的一块玻璃，用蚀刻成它的统治者）。测量在一个1平方毫米的像素数框使用的计算机程序。 注意:关键此协议的重复性和标准化很大程度上取决于1mm 2中框被计数的正确放置。 </li><li>使用对比度增强工具变暗毛细血管，减轻的背景下，这将最大限度地提高毛细血管的可视化。毛细管从背景的初始分化为在后续步骤中的图像的适当的裁切重要。通过调整使用的图像做这个要做到这一点，点击"最适合的直方图。":捕捉，亮度，图像的直方图，最适合的。 </li><li>作物感兴趣区域（ROI）的毛细管计数为新图像的区域（选择，感兴趣区域）。使用1毫米2中，这是通过校准图像530的像素等于1毫米来确定。检查该裁剪出的图像放置毛细血管袢的顶点在图像的最顶端。 </li><li>拼合因此未来所有的图像调整将均匀地涂在图像图片。要做到这一点点击:进程选项卡，2D过滤器，压平，"光明"，对象宽度BG强度为"75"，适用。 </li><li>提高图像的对比度，因此毛细管最大限度可视化。要做到这一点点击:调整选项卡，显示屏，对比度提升到75。 </li><li>去斑的图片，点击进程选项卡，2D过滤器，去斑，内核尺寸7×7平滑毛细血管的边缘，适用。 </li><li>最终确定的图像对比度使毛细血管是黑色的，背景是白色的。执行此步骤通过调整直方图的"最适合"的模式。 注: 请参考图2代表结果的处理图像应该是什么样子以下步骤的一个例子。 </li></ol> 2.执行毛细管计数/定性测定毛细血管密度<ol><li>每个图像，手动选择使用"目标对象"featur一个明确定义的毛细管中的一个部分E要被识别为对象的程序计算。然后，选择的背景的一小部分，采用了"背景"的功能，作为参考，以需要由所述计数算法被忽略的区域。 注意:这些亮点的组合导致而忽视了背景噪音所有毛细管加以强调。定量（计数）协议使用的计算机的功能来区分基于颜色和形态图像的某些部分。 </li><li>使用计数功能能够即时计算所有毛细管与成像设备的形象。为了避免计算的背景噪声如毛细血管设置计算的对象到5个像素的最小直径。 </li><li>对于每个单独的，计数为3的平均 - 4个图像，以获得一个更可靠的评估。 注: 请参考图3。代表结果的形象应该是什么样子的计算过程中的一个例子。 </li></ol&gt; 3.创建和使用宏来自动图像处理注:为了节省时间，宏可以以一个或多个图像自动执行的过程特定顺序创建。这些序列可以定制，以使该图像修改更快。从本质上说，这些宏记住如何图像进行处理，并迅速和没有用户输入执行所有的步骤。 12毛细管图像执行计数需要20至30分钟之间的这种实验室用宏（2至3每个画面分钟），相对于每个图象约8分钟而不宏。因此，使用宏是比手动处理每个单独的图像3至5倍更有效的。 <ol><li>为了创建宏，选择"记录宏"，并在一个图像执行的步骤和过程所需的，如在步骤1和2如上所述。在此基础上的图像处理步骤进行以供将来参考进行命名宏。当USI纳克未来图片宏，只需单击"运行宏"，程序将自动应用所记录的增强功能需要的图像（S）。 注:本实验使用宏来执行的在几秒钟的方法第1节所有，但其中一个步骤。需要用户输入唯一的步骤是选择在哪里裁剪图像，步骤1.2。 </li></ol>

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The authors have no conflicts of interest.

甲襞capillaroscopy表明承诺作为一个在未来各种肿瘤，心血管疾病，风湿病和疾病临床上的应用程序非常有用的工具。图像采集过程是研究人员之间相当一致的，但目前有多种方法用于图像处理和分析。方法目前包括计算机及人工毛细血管计数。手动计数是有问题的，因为它们是费时，并受到用户的主观性和疲劳。当前的计算机为基础的方法需要用户输入的一个高层次，无论在图像增强处理和量化处理。这里描述的新方法需要相对较少的用户培训或参与，所述图像增强步骤是完全自动化的。用户输入仅需要计数过程最初从背景区别毛细管中的处理的图像。使用自动宏所以此处所述的三到五倍比马努更有效盟友处理每幅图像。 可靠的标准化计算机算法来定量capillaroscopy数据缺乏。 为了减少主观性，提高效率的需要毛细管定量可靠的标准化计算机为基础的方法。这里所描述的技术展示与这两个区域内和跨观察员重复性为0.93至0.98内相关的好成绩出色的可靠性。之前我们已经报道通过电脑毛细血管密度评估得到的结果的相关性，相对于人工计数的黄金标准。基线，缺血后，并用软件来完成静脉充血数量和相应的人工计数之间21皮尔逊相关性在10个受试者0.78，0.78，0.71（均P &lt;0.05），说明两种方法之间合理的协议。 该实验室的形象操作步骤利用了若干计算机处理工具。步骤1.3，图像"压扁"，除去各种"层"中存在的每个图像。这首先必须这样做将来所有的图像处理程序将被均匀地应用到图像的所有部分。对比度调节双方加深毛细血管和相形见绌出的背景下，因此使毛细血管更加明显。的"去散斑"过程平滑毛细管的边缘，同时保持其尺寸和形状。虽然似乎是在一个除斑的图像肉眼无差异，这是为了确保多毛细血管不会在计票过程融合在一起的一个重要过程。最终确定使用"最适合直方图"的形象排除所有像素在两个极端的直方图。这有助于限定毛细管的边界，同时进一步提高毛细血管和背景之间的对比度。总体有3对比度增强步骤，并且所有三个需要最大程度的毛细管数量最终图像的清晰度。 偶尔，该计划将依靠过多或过少的毛细血管。解决这个问题的第一步是要撤消高亮，只是再次尝试高亮处理。如果毛细管是被强调的不当，调整最低毛细管直径可能是必要的。作者建议的5个像素，默认最小直径。如果该程序计数太多毛细管或计数一个毛细管作为多个毛细管用户应该由一个或两个像素增加的最小直径。另一方面，如果程序不计算暗像素团的毛细管时，用户可以通过一个像素减少的最小直径。 还有一个需要统一的甲床内的这些计数的位置上。看到的表中，对在该同一个体是高度位置相关的，不同而异大大根据的甲床的一部分正在计数。 关键步骤协议包括毛细血管的正常和最佳的可视化。允许毛细管在这个协议中的最佳可视化的步骤是完全自动化，从而允许快速，准确的图像处理。虽然这些方法代表了可靠性的一个重大进步，缓解处理和计数capillaroscopic图像的，该技术的主要限制是半自动化的计数处理。理想的是，一个完全自动化的过程将在不久的将来被创建。研究人员应该感到鼓舞，以建立在本文中，以建立一个完全自动化的临床上有用的技术，可以让患者的甲襞毛细血管密度的快速定量描述的方法。

微血管成像是一个快速增长的领域有许多潜在的临床应用价值。2例如 ，肿瘤学家使用微血管成像，以确定肿瘤血管生成的程度，产生关于肿瘤和洞察力的状态变成可能的治疗选择有价值的信息。3 4然而，甲襞capillaroscopy也许是微血管成像的最具成本效益和广泛适用的形式。研究人员正在利用视频甲襞capillaroscopy研究血流量和调查毛细血管形态。5 6视频和静态图像甲襞capillaroscopy的附属品照顾诊断和治疗雷诺氏现象和各种结缔组织病如系统性硬化症。2 甲襞capillaroscopy具有多种潜在的心血管应用程序。目前的研究使用甲襞capillaroscopy建议该糖尿病类型1和类型2的患者表现出异常毛细管形态的高患病率，但有不变的毛细管密度相比，非糖尿病个体7-8 Capillaroscopy也进行了研究实验中高血压。结构毛细管稀疏导致降低的毛细血管密度已被证明在高血压个体相比，非高血压的个体。9-10相对于这些老年高血压患者（平均年龄40以上）谁展示稀疏结构，最近的研究已经证明，年轻的高血压患者（平均年龄40岁）有没有结构疏松功能稀疏。11这表明，功能性稀薄之前发生，并且随着时间的推移可能会发展为结构疏松。 有趣的是，高血压患者与特定的降压药如培哚普利治疗/吲达帕胺显示规范人毛细血管密度及治疗后血管内皮功能，而与ACE（血管紧张素转化酶）抑制剂或利尿剂治疗维持，尽管可比控制血压低的毛细血管密度。12这表明，一些抗高血压药物可能会恢复正常毛细血管密度扭转毛细管稀疏由高血压引起的。此外，其他研究人员已经表明，在食盐摄入量的减少导致逆转高血压个体功能和结构的毛细管稀薄。13 尽管这种技术的各种潜在的临床应用中，很少有标准化技术定量毛细血管密度的图像。2迄今为止，研究人员已经发现，毛细血管密度的结果是从两帧内观察者和观察者间的角度仅当精确重现的同一地区被每次计数。1,14 15 </sup&gt;值得注意的是，以往的研究人员已经基本上完成毛细血管计数手动使用肉眼，9 16 17 18这是一个缓慢而主观的过程。 标准化，基于计算机算法毛细血管图像的定量理论提供更加高效和可重复的数据分析，用更少的主体性，促进capillaroscopy的临床应用。一些研究人员已经确实使用基于计算机的程序，以从甲襞capillaroscopic图片定量的数据。1,6 19 20然而，只有一个出版迄今为止描述了一种复杂的软件程序的可用的可靠性定量capillaroscopy数据，1，并将该软件是复杂的作为先前在上文所指出的要求来算完全相同的视野。在这里，我们提出了一个简单的，可靠的协议，使用一个标准化的算法允许定量毛细管使用多个视场。使用多个视野，不仅简化了程序，而且还允许正常生物变化的毛细管计数的评估。 这项研究的目的是描述一个基于可重复和高效的计算机算法，它规范了毛细管定量的过程。虽然这些方法都没有完全自动化它们需要非常少的用户输入，并且提供图片的快速，可靠的定量。

<table><tbody><tr><td>Image-Pro Premier</td><td>Media Cybernetics, Inc</td><td>9.1</td><td>Image processing software</td></tr></tbody></table>

a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy

Capillaroscopy为直接可视化的微循环的非侵入性的，高效的，相对便宜和容易学习方法学。的capillaroscopy技术可以提供深入了解患者的微血管健康，导致各种潜在的有价值的皮肤病，眼科，风湿病和心血管临床应用。此外，肿瘤生长可能是依赖于血管生成，这可以通过测量肿瘤内微血管密度进行定量。然而，目前还几乎没有标准化的技术，只有一个出版迄今为止报告的当前可用的，复杂的基于计算机的算法的可靠性为定量capillaroscopy数据。1本文介绍了一种新的，更简单的，可靠的，标准化的毛细管计数算法对于定量甲襞capillaroscopy数据。一个简单的，可重复的电脑capillaroscopy算法，因为这将有利于更多广泛使用，研究人员和临床医生的技术。许多研究者目前正在分析capillaroscopy图像手工，促进成果的使用者的疲劳和主观性。本文介绍了一种新的，易于使用的自动除可重复的，半自动化计数算法的图像处理算法。这个算法使图像的分析中，同时降低主观性分钟;的训练时间仅有极少量（在我们的经验，不到1小时），需要学习的技术。

该图像处理程序的目标是从背景图像区分的毛细血管，这样他们可以被精确定量。这两个不完整的图像处理和过度的图像处理，损害程序的量化毛细血管能力。 正如图3中，不完整的图像处理使得毛细管难以从背景区别开来。该用户能够容易地辨别一个毛细管的边界，因为上述的计数方法取决于用户的准确强调几个毛细管能力是至关重要的。另一方面， 如在图3看到的，不必要的图像处理步骤，应用程序可以导致模糊的毛细血管，因此也可能是有害的量化过程。 一个最佳处理后的图像可在30秒进行计数，并且清楚地从背景中区分毛细血管作为以及从彼此作为单个毛细管。处理后的图像的一个例子可见图2的D部分中， 与图3所示的计数图像。 毛细血管密度不同，取决于甲床的位置被计算。 表1表明，毛细血管密度与来自毛细管在甲床顶行距离的增加。投资回报率安置标准化是关键计数的重现性。 图1显示了图像可以与不同的投资回报安置被改变。 <table><tbody><tr><td> ID </td><td> T1 </td><td> T2 </td><td> T3 </td><td> T4 </td><td>平均顶部</td><td> M1 </td><td> M2 </td><td> M3 </td><td> M4 </td><td>平均中东</td><td> L1 </td><td> L2 </td><td> L3 </td><td> L4 </td><td>最低</td></tr><tr><td>患者A </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>底线</td><td> 46 </td><td> 45 </td><td> 44 </td><td> 46 </td><td> 45.25 </td><td> 64 </td><td> 62 </td><td> 62 </td><td> 62 </td><td> 62.5 </td><td> 66 </td><td> 67 </td><td> 66 </td><td> 66 </td><td> 66.25 </td></tr><tr><td>静脉闭塞</td><td> 51 </td><td> 53 </td><td> 49 </td><td> 59 </td><td> 53 </td><td> 59 </td><td> 61 </td><td> 64 </td><td> 69 </td><td> 63.25 </td><td> 70 </td><td> 70 </td><td> 75 </td><td> 72 </td><td> 71.75 </td></tr><tr><td>患者乙</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>底线</td><td> 47 </td><td> 51 </td><td> 48 </td><td> 51 </td><td> 49.25 </td><td>73 </td><td> 74 </td><td> 75 </td><td> 81 </td><td> 75.75 </td><td> 76 </td><td> 85 </td><td> 81 </td><td> 80 </td><td> 80.5 </td></tr><tr><td>静脉闭塞</td><td> 68 </td><td> 57 </td><td> 65 </td><td> 64 </td><td> 63.5 </td><td> 75 </td><td> 78 </td><td> 76 </td><td> 72 </td><td> 75.25 </td><td> 91 </td><td> 89 </td><td> 93 </td><td> 83 </td><td> 89 </td></tr><tr><td>病人Ç </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>底线</td><td> 51 </td><td> 54 </td><td> 51 </td><td> 56 </td><td> 53 </td><td> 66 </td><td> 59 </td><td> 58 </td><td> 60 </td><td> 60.75 </td><td> 60 </td><td> 61 </td><td> 62 </td><td> 69 </td><td> 63 </td></tr><tr><td>静脉闭塞</td><td> 62 </td><td> 66 </td><td>五7 </td><td> 59 </td><td> 61 </td><td> 63 </td><td> 63 </td><td> 73 </td><td> 65 </td><td> 66 </td><td> 83 </td><td> 74 </td><td> 81 </td><td> 77 </td><td> 78.75 </td></tr></tbody></table> 表1:变异毛细管计数与差分定位在指甲床此表显示了三种不同的患者（A，B，C），当ROI框可变位于顶部（计数T1 - T4）获得的计数，中间（M1 - M4）和下部区域（L1 - L4）的指甲床。的平均计数从顶部增加，降低区域，表明需要的ROI框放置标准化比较来自不同实验室获得的计数。 在步骤1.2中描述的区域进行计数，基线计数的范围应该是30至60的毛细管/毫米2，而静脉闭塞计数范围可在任何地方从50到100 如表1，这些ðensities从其他文献中有所不同。在作者实验室得到毛细血管密度计数是最有可能更低，因为此实验开始计数在毛细管的第一行，这里的密度是最低的。 如表1所示，在甲床的下部区域进行计数增加由安东尼奥斯等人 9和Debbabi先前获得的朝向值的计数等人的 16这一差异说明通过计数所述第一需要标准化在甲床capillaroscopy定量（最近）排毛细血管。计数在毛细管的第一行也是最优的，因为毛细管是最清楚，完整地显现在第一行中，并逐渐成为与随后的行不太显眼。 使用N = 10受试者和两个独立的观察员蒙蔽重复性研究进行。可靠性是指结果的平均A，B和C计数，通过AV获得<html> eraging跨4图像每个结果。在A，B和C的计数表示用于评估微血管健康，简要总结同一个体中的不同生理状态。详情已先前公布的21。毛细血管密度被定义为每毛细血管的手指甲襞皮肤平方毫米（mm 2）的数量。 A阶段是一个休息的基线阶段，毛细血管被连续灌注16。 B阶段期间postocclusive反应性充血发生。这些计数代表连续灌注和间歇性灌注（功能储备）的毛细血管的总和。这个阶段被用作毛细管功能 16的量度。 C阶段发生静脉闭塞期间，因此表示最大毛细血管密度包括灌注（有效的红血细胞（RBC）的运动）和nonperfused（填充有停滞，非移动红细胞）毛细血管。22 内容】"&gt;对于内部信度，组内相关（ICC）为0.93的平均值的统计，0.93的平均乙计数和0.94的均值C计数。对于间信度的相关系数均0.94的平均值的数量， 0.98平均乙计数和0.94的均值C重要的。因此，这里所描述的技术展示与这两个区域内和跨观察员重复性好成绩出色的可靠性。 <img alt="图1" src="/files/ftp_upload/53088/53088fig1.jpg" /> 图1.规范的裁剪位置。这个数字说明了ROI框的位置变化明显改变裁剪后的图像。在左边，方块被放置过低，切断毛细管的第一行。中间框置于过高，引起毛细管的第一行上方的空白空间中。右边的框中最佳放置。它的裁剪图像显示C的第一行apillaries在图像的最顶端。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53088/53088fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53088/53088fig2.jpg" /> 图2.阶段图像处理的（A）的 A阶段显示从被检者的甲床使用单色相机拍摄初始图像;（B）的阶段B表示第一对比增强后的原始图像。绿色框显示一个530 x 530像素的盒子，相当于1×1毫米框的摄像头;（三）C阶段表示从图像B 1mm的框裁剪;（四）D阶段显示了应用后，提高了图像上面讨论的增强。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53088/53088fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </p&gt; <img alt="图3" src="/files/ftp_upload/53088/53088fig3.jpg" /> 图3.最终计数形象的提升，计算图像。确定这个图片总数为54毛细血管/毫米2。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53088/53088fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53088/53088fig4.jpg" /> 图4.不正确的图像处理。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53088/53088fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 左侧的照片显示了未加工的足够的照片。毛细血管是很难从背景区分和定量过程将是负affec特德。右边的照片显示不正确的图像处理后，相同的图像。单个毛细管难以从他们的邻居区分开，从而定量过程中会受到负面影响。

Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

对于毛细血管密度的定量使用甲襞Capillaroscopy可再生的计算机化方法

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

a-reproducible-computerized-method-for-quantitation-capillary-density

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.9K 次观看.  Thomas Jefferson University. Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm. 

视频: A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的光学显微镜：测量细胞生物物理特性与标准光学显微镜

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

科研

生物工程

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

非侵入性的评估微血管和内皮功能

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

医学

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

生物化学

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

生物学

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

漫反射光谱分析: 在拇指下进行毛细管填充试验

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

神经科学

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

非增殖性和增殖性视网膜病变小鼠整个视网膜血管参数的定量

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

评估毛细血管和其他血管对光学相干断层扫描血管造影测量的黄斑灌注密度的贡献

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

自动定量分析细胞计数程序使用ImageJ插件

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...