作者没有致谢。

所报告的方法被用于一项研究, 它得到了伯恩茅斯大学研究伦理学委员会2016年2月9日的伦理批准 (参考文献 10571). 
 1. LSCI 设置 <ol> <li> 将 moorFLPI LSCI 后面板连接到其三插槽 (电源、通用串行总线 (USB) 和 IEEE1394), 以便系统正常工作. </li>
	<li> 使用4螺钉将桌面支持臂装配到 moorFLPI LSCI 翻转, 并固定在支撑臂上. </li>
	<li> 旋转安装托架以便在连接时向下成像. 
	注: LSCI 有三控制: (1) 变焦调节器和 #8211; 随着成像器设置在一个位置, 较小的放大倍数可以分别调整为较小和更大的视野的最大和最小设置。为确保重复性, 提供了一个索引环标签;(2) 焦点调节器和 #8211; 这取决于测量距离, 必须在设置图像位置后进行调整。为确保重复性, 提供了一个索引环标签;和 (3) 偏光片和 #8211; 线性偏振滤波器可使曝光的机构和 #8211 的镜面反射最小化; 旋转安装可通过360和 #176 进行翻转;. </li>
	<li> 安装软件来控制摄像头。该软件分为两个模块, 提供了一个测量和审查功能. </li>
</ol> 2。参与者准备 <ol> <li> 确保在温度控制室 (22 和 #177; 1 和 #176; C) 中执行评估, 并且参与者在测试前10分钟就位, 以适应室温. </li>
	<li> 避免强烈的人工光源和阳光照射在参与者或 LSCI 上, 因为环境光线会影响 moorFLPI 近红外激光源的运行在 785 nm. 
	注: 一个简单的测试, 以确定是否可以接受的环境光线水平是通过打开成像设置窗口和阻碍激光。如果图像是几乎完全黑色的, 那么不需要进一步的步骤;如果仍然存在太多的环境光, 则需要进一步的操作. </li>
	<li> 确保参与者在整个评估过程中都能轻松自如, 如果坐在地上, 并避免对话. </li>
	<li> 放置 8 cm 2 粘附不透明贴片 (AOP) (如 、Leukotape) 在皮肤区域上以遮盖血流。这是为了解释 LSCI 的缺点, 在高灵敏度的工件运动, 和信号后向散射将用于测量微循环血流. </li>
</ol> 3。微循环图像测量 <ol> <li> 选择和 #39; 空间处理和 #39; 每秒捕获25帧, 以 152 x 113 像素为单位. </li>
	<li> 选择和 #39; 实时图像测量和 #39; 调整成像器20厘米以外的位置, 然后调整缩放、焦点和偏光镜以最小的镜面反射。图像应该显得相当和 #39; 平面和 #39; 和无特征. </li>
	<li> 设置20毫秒的曝光时间, 以对小的变化和低通量进行高灵敏度. </li>
	<li> 使用25赫兹的显示速率和0.3 秒的时间常数来解释快速的血流变化, 并通过降低图像噪声来达到最佳对比度. </li>
	<li> 创建两个相等的大小 (2 cm 2 ) 感兴趣的区域 (roi), 称为 roi 1 和 roi 2。使 ROI 2 对齐, 因此它位于 8 cm 2 AOP 中。小心, 使 roi 不交换, 但保持接近2-4 厘米, 以减少 re-centering 的需要, 如果任何机械运动导致 ROI 2 不再在 AO 地区. 
	注意: 在低强度和高强度的区域, 血流测量将不太准确, 所以有一个最佳增益设置是很重要的。增益值范围介于 0-200 之间。在 70-80 范围内实现了最佳增益设定值. </li>
	<li> 相对于参照基线执行流量测量。在这种方法的情况下, 引入和 #39; 休息和 #39; 阶段作为参考基线。因此, 表达 #39; 快速和 #39; 和 #39; 慢速和 #39; 从基线、#39、休息和 #39, 阶段的血流百分比变化; 阶段. </li>
	<li> 以视频格式记录血流测量, 并使用图像复查模块保存离线分析. </li>
</ol> 4。脱机分析 注意: moorFLPI 图像审阅软件允许打开视频进行分析. 
<ol> <li> 在一系列的平均血流记录后, 计算 roi 内的平均通量。roi 1 是对皮肤血流的真实测量, roi 2 是来自 AOP 的散射激光散斑皮肤信号. </li>
	<li> 通过从 roi 1 (皮肤血流) 中减去 roi 2 来计算平均血流. 
	roi 1-roi 2 = 平均血流 </li> </ol>

<ol>
	<li>Mahe, G., Rousseau, P., Durand, S., Leftheriotis, G., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contract+imaging+accurately+measures+blood+flow+over+moving+skin+surfaces.">Laser speckle contract imaging accurately measures blood flow over moving skin surfaces.</a> Microvas Res. 81 (2), 183-188 (2010).</li><li>Rousseau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+&amp;#34;region+of+interest&amp;#34;+and+&amp;#34;time+of+interest&amp;#34;,+both+reduce+the+variability+of+blood+flow+measurements+using+laser+speckle+contrast+imaging.">Increasing the &amp;#34;region of interest&amp;#34; and &amp;#34;time of interest&amp;#34;, both reduce the variability of blood flow measurements using laser speckle contrast imaging.</a> Microvas Res. 82, 88-91 (2011).</li><li>Hecht, N., Woitzik, J., Dreier, J., Vajkoczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraoperative+monitoring+of+cerebral+blood+flow+by+laser+speckle+contrast+analysis.">Intraoperative monitoring of cerebral blood flow by laser speckle contrast analysis.</a> Neurosurg Focus. 27 (4), 1-6 (2009).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Leftheriotis, G., Abrham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+experimental+conditions+on+noncontact+laser+recordings+in+microvascular+studies.">Impact of experimental conditions on noncontact laser recordings in microvascular studies.</a> Microcirculation. 19 (8), 669-675 (2012).</li><li>Cheng, H., Duong, T. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+laser-speckle-imaging+analysis+method+and+its+application+to+retinal+blood+flow+imaging.">Simplified laser-speckle-imaging analysis method and its application to retinal blood flow imaging.</a> Opt Lett. 32 (15), 2188-2190 (2007).</li><li>Doherty, J., McNamara, P., Clancy, N. T., Enfield, J. G., Leahy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+instruments+for+investigation+of+microcirculatory+blood+flow+and+red+blood+cell+concentration.">Comparison of instruments for investigation of microcirculatory blood flow and red blood cell concentration.</a> J Biomed Opt. 14 (3), 034025 (2009).</li><li>Briers, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+speckle+contrast+imaging+for+measuring+blood+flow.">Laser speckle contrast imaging for measuring blood flow.</a> Opt Appl. 37 (1), 139-152 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> moorFLPI User Manual. (8), (2012).</li><li>Mahe, G., Durand, S., Humeau-Heurtier, A., Abraham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Experimental+Conditions+on+Noncontact+Laser+Recordings+in+Microvascular+Studies.">Impact of Experimental Conditions on Noncontact Laser Recordings in Microvascular Studies.</a> Microcirculation. 19, 669-675 (2012).</li><li>Bahadori, S., Immins, T., Wainwright, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+calf+neuromuscular+electrical+stimulation+and+intermittent+pneumatic+compression+on+thigh+microcirculation.">The effect of calf neuromuscular electrical stimulation and intermittent pneumatic compression on thigh microcirculation.</a> Micro Res. 111, 37-41 (2017).</li><li>Omarjee, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimisation+of+movement+detection+and+artifact+removal+during+laser+speckle+contrast+imaging.">Optimisation of movement detection and artifact removal during laser speckle contrast imaging.</a> Miscrovas Res. 97 (1), 75-80 (2015).</li></ol>

作者没有什么可透露的。

本研究的目的是介绍一种新的方法来测量和分析的交替速度的血液微循环在一个单一的实验使用 LSCI。测量可以受到环境光、振动和参与运动的影响, 包括呼吸和抽搐。该协议中概述的步骤都是为了尽量减少这些影响, 并获得可靠和可重复的血液微循环测量。
人们强调, 该协议中的每一个步骤对于准确测量血液微循环都是至关重要的, 在对所有可能的设置选项和组合进行顺序测试后发现了该技术, 包括:时间常数, 变焦设置, 曝光时间, 显示率, 增益和通量图像调色板。结果进行了分析和重复使用现场视频显示和离线分析找到最佳的成像设置。这是必不可少的, 因为图像处理软件使用的事实, 高灌注产生的激光散斑模式的快速变化, 并因此在视频图像中产生了一个低对比度清晰的散斑区。灌注图像, 然后创建在一个 color-coded 的微循环灌注图。
实验区和参与准备是必不可少的, 这可以通过避免工作附近的日光源 (窗口) 或强光源的人工光, 因为这些可能会干扰 moorFLPI 附近的红外激光源。该协议还介绍了一个 AOP, 因为它被承认, 环境振动和参与者的运动都产生的信号是无法区分的血流。AOP 证明是一个简单而有效的选择, 提供了一个薄但不透明, 光和容易接近的选项, 有一个微观粗糙的表面积, 以避免显着镜面反射。Omarjee et al.的初步研究11突出显示了一个潜在的限制, 即 Leukotape 创建的反射信号振幅与皮肤不同, 并且各主体之间的差异很大;然而马希et al.1发现参与者之间没有显著差异。虽然 Leukotape 比其他定制的双层粘接片更易访问, 但通过使用另一种 AOP, 可以提高方程 (1) 测量的精确度。
离线分析部分强调了 roi 大小的重要性, 以及它们在感兴趣区域的位置。最初, 一个更大的 roi 1 (大约 8 cm2) 被尝试, 其中覆盖了 roi 2。这种方法很快成为 un-reliable 由于工件运动导致的 roi 2 移动和现场实验必须停止, 以重新的 roi 2。另外一个短期的原因是, 由于 ROI 1 覆盖了 aop, 平均通量不再将 aop 下的区域作为一个帐户, 因为不再有散射信号。这意味着大量的血液微循环被忽视, 因此产生的通量数据是不正确的。因此, 一种方法, 其中两个 roi 2 cm2, 具有 8 cm2的 AOP, 在 roi 1 和 roi 2 之间没有交互 (但在彼此之间保持2-4 厘米), 为血液测量提供了可靠且可重复的分析技术。微循环.

激光散斑造影成像 (LSCI) 是一种行之有效的非接触、实时监测方法, 用于分析血流微循环1,2,3,45,6,7. 本文所用的 LSCI moorFLPI 全域。利用一种称为 "激光散斑" 的现象测量大面积高时空分辨率的血流灌注是该装置6的主要优点之一。real-time 评估的微循环是通过拍摄的模式, 通过相机使用散斑模式。鉴于 moorFLPI LSCI 用于临床和生理学研究, 图像处理软件的工作原理是, 高灌注产生的激光散斑模式的快速变化, 然后由带电耦合器件 (CCD) 集成生成低对比度的区域8。对比度是量化的, 由此产生的流量是彩色编码产生一个灌注图像8。
不幸的是, LSCI 是非常敏感的环境振动, 工件和运动的主题领域9。到目前为止, 这为交流流状态的研究提供了挑战。本文详细介绍了显式技术概述了在最近的研究10 , 其中一个神经肌肉电刺激装置被用来测量血液微循环时, 有肢体的运动正在检查。

<table><tbody><tr><td>moorFLPI LSCI</td><td>Moor Instruments</td><td>Not Available - Online Link Provided in descreption</td><td>moorFLPI is an instrument designed for the measurment of blood flow within microvasculature by using infra red laser speckle contrast analysis.&amp;nbsp; https://gb.moor.co.uk/</td></tr><tr><td>moorFLPI Image Review Module</td><td>Moor Instruments</td><td>No Available - Online Link Provided</td><td>Used with moorFLPI, user can record and measure changes in blood flow by changerating a colour coded map of tissue perfusion.&amp;nbsp; https://gb.moor.co.uk/</td></tr><tr><td>Leukotape</td><td>BSN Medical</td><td>72978-10</td><td>Medical tape with microporous surface. http://www.bsnmedical.co.uk/fileadmin/z-countries/United_Kingdom/PDF/L/Leukotape_K_A46PP_low_res_11112013.pdf</td></tr></tbody></table>

a novel approach to overcome movement artifact when using a laser speckle contrast imaging system for alternating speeds of blood microcirculation

激光散斑造影成像仪 (LSCI) 提供了一种强大而简单的测量微循环血流的技术。理想的血液动力反应, LSCI 的使用方式与传统的激光多普勒成像仪 (LDI) 相同。然而, 最大的皮肤深度约1毫米, LSCI 设计的重点主要是表面血流。它用于测量 15 cm x 20 cm 的皮肤表面积。本文介绍的新技术是微循环的交替速度;即使用 LSCI 的慢速和快速流量测量。该技术还克服了 LSCI 最大的缺点, 即对工件运动的灵敏度高。通过从激光散斑皮肤信号中减去来自 aop 的 LSCI 信号, 引入了一种粘着不透明贴片 (aop) 来满足微循环血流的满意记录。最佳的设置也被定义了, 因为 LSCI 是最强有力的, 当通量变动测量相对于参考基线, 以血液微循环通量表示作为一个百分比变动从基线。这些变化可用于分析血流系统的状态。

在图 1中概述了 LSCI 实验设置, 并确定了功能工具。一个典型的参与者准备测量的血流量在前大腿区域的说明。可调式安装支架允许旋转 LSCI 的血液流量测量在任何特定的皮肤区域微血管。图 2概述了典型的原始散斑图像和转换的散斑图像的示例, 并在《血液微循环测量协议》中列出了定制的设置。
图 3显示了 AOP 的皮肤区域和位置的示例 (步骤 3.1), LSCI 原始成像设置 (步骤 3.2), 一个慢血流的实时图像 (步骤 3.3) 和一个快速血流的实时图像 (步骤 3.4) 实现了在连续的数据记录的交替通量美国moorFLPI LSCI调色板 color-coding 设置允许差异之间的流量水平。与标准调色板的16种颜色, 低通量被视为蓝色, 中通量值被视为绿色和高通量值被视为橙色和红色。
皮肤血流是用激光散斑灌注单元 (LSPU) 来表达的。图 4显示了一个皮肤区域的 roi 1、roi 2 和 AOP 的图形表示形式。在离线分析中, 使用 roi 1 和 roi 2 的数据和公式 (1) 计算平均血流。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56415/56415fig1.jpg"> 
图 1: moorFLPI LSCI 实验设置.台式机支持臂、电缆输出、位置控制 (变焦调节器、聚焦调节器和偏振器)、AOP 和膝上型计算机用于配置图像设置。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56415/56415fig2.jpg"> 
图 2: 数据记录前的原始散斑图像2.1-2.2) 流量和原始散斑图像的配置糟糕, 导致高增益, 能见度差, 这将导致一个不准确的血液流量测量。2.3-2.4) 系统配置为按协议, 导致正确的增益和最大的能见度可靠的结果。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56415/56415fig3.jpg"> 
图 3: 使用 moorFLPI LSCI 设置和记录测量的概述.3.1. 具有 2 cm2 AOP 的皮肤 (大腿) 区域, 用于计算工件移动。3.2) 一个原始散斑 ' 平面和无特征 ' 图像表明良好的背向散射光强的最佳设置。3.3) 血流缓慢的实时图像记录。3.4) 一个快速血流的实时图像记录。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56415/56415fig4.jpg"> 
图 4: roi 1 的图形表示、roi 2 和在皮肤区域上的 AOP 布局.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">休息 (基线参考) (LSPU)</td>
			<td colspan="4">慢血流量 (LSPU)</td>
			<td colspan="4">适度血流 (LSPU)</td>
			<td colspan="4">快速血流 (LSPU)</td>
		</tr>
		<tr>
			<td>平均通量-ROI 1</td>
			<td>平均通量-ROI 2</td>
			<td>平均血流</td>
			<td>平均通量-ROI 1</td>
			<td>平均通量-ROI 2</td>
			<td>平均血流</td>
			<td>血流量从基线增加%</td>
			<td>平均通量-ROI 1</td>
			<td>平均通量-ROI 2</td>
			<td>平均血流</td>
			<td>血流量从基线增加%</td>
			<td>平均通量-ROI 1</td>
			<td>平均通量-ROI 2</td>
			<td>平均血流</td>
			<td>血流量从基线增加%</td>
		</tr>
		<tr>
			<td>157。9</td>
			<td>35。1</td>
			<td>122。8</td>
			<td>178。5</td>
			<td>41。6</td>
			<td>136。9</td>
			<td>10。9</td>
			<td>216。9</td>
			<td>44。6</td>
			<td>172。3</td>
			<td>33。5</td>
			<td>418。9</td>
			<td>77。5</td>
			<td>341。4</td>
			<td>94。2</td>
		</tr>
	</tbody>
</table>
表 1: LSPU 中的平均通量, 用于 roi 1 和 roi 2 的基线、慢速、中度和快速血流.血流的增加是表示从基线阶段的百分比变化。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56415/56415fig5.jpg"> 
图 5: roi 1 和 roi 2 在皮肤表面积 (大腿) 上定位的示例.16色调色板概述了显影的克里斯·赖维斯。数据记录的 roi 1 和 roi 2 在灌注单元和减去如解释的公式1的血液微循环的测量。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56415/56415fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation at JoVE.com

A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

用激光散斑对比成像系统克服运动伪影的新方法血液微循环的交替速度

本研究介绍了一种利用激光散斑造影仪测量和分析血液微循环交替速度的新方法。

The laser speckle contrast imager (LSCI) provides a powerful yet simple technique for measuring microcirculatory blood flow. Ideal for blood dynamic ...

a-novel-approach-to-overcome-movement-artifact-when-using-laser

Nanyang Technological University

Research

JoVE Journal

Medicine

8.4K 次观看.  Bournemouth University. 本研究介绍了一种利用激光散斑造影仪测量和分析血液微循环交替速度的新方法。

视频: A Novel Approach to Overcome Movement Artifact When Using a Laser Speckle Contrast Imaging System for Alternating Speeds of Blood Microcirculation

How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow. The technique analyzes fluctuations in a dynamic speckle pattern to detect the movement of particles similar to how laser Doppler analyzes frequency shifts to determine particle speed. Because it can be used to monitor the movement of red blood cells, LSCI has become a popular tool for measuring blood flow in tissues such as the retina, skin, and brain. It has become especially useful in neuroscience where blood flow changes during physiological events like functional activation, stroke, and spreading depolarization can be quantified. LSCI is also attractive because it provides excellent spatial and temporal resolution while using inexpensive instrumentation that can easily be combined with other imaging modalities. Here we show how to build a LSCI setup and demonstrate its ability to monitor blood flow changes in the brain during an animal experiment.

How to Build a Laser Speckle Contrast Imaging LSCI System to Monitor Blood Flow

This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow.

如何建立一个激光散斑对比度成像（LSCI）系统，以监测血流

Laser Speckle Contrast Imaging (LSCI) is a simple yet powerful technique that is used for full-field imaging of blood flow.  The technique analyzes ...

科研

神经科学

Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to give an accurate velocity profile. A protocol is presented for &mu;PIV measurements of blood flows in microchannels. At the scale of the microcirculation, blood cannot be considered a homogeneous fluid, as it is a suspension of flexible particles suspended in plasma, a Newtonian fluid. Shear rate, maximum velocity, velocity profile shape, and flow rate can be derived from these measurements. Several key parameters such as focal depth, particle concentration, and system compliance, are presented in order to ensure accurate, useful data along with examples and representative results for various hematocrits and flow conditions.

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.

微观粒子图像测速技术，微血液流动的速度剖面测量

Micro-particle image velocimetry (&mu;PIV) is used to visualize paired images of micro particles seeded in blood flows. The images are cross-correlated to ...

生物工程

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

微流控控制环境的红细胞聚集动态调查

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染:肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

免疫与感染

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

微血管中场流的空间时间分析

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

医学

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

一种监测人牙龈移植新生血管的新方法

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

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