我们要感谢什雷亚·沙阿的校对和建议。

1. 多普勒幻影准备设置 （图 2A）
<ol><li>将腹腔泵、血液模拟液库、脉搏阻尼器和多普勒流幻影与塑料管连接起来。</li>
	<li>选择直径为 4 mm 的运河。</li>
	<li>将泵编程为弹出 720 mL/min 的液体 0.3 秒，然后弹出 50 mL/min 的 0.7 秒，分别模拟晶体和硅藻心脏相位</li>
	<li>运行泵并轻轻摇动管道以排出潜在的气泡。 注意：操作员可以选择不同的运河直径和不同的泵速率，但必须确保超声波序列足够快，以获得最快的流速。稍后提交的 Eq. 3 有助于设计序列。</li>
</ol>2. 超高速超声波扫描仪设置 （图 2A）
<ol><li>将支持超快的研究扫描仪与主机连接起来，并连接 PCI 快速链接。</li>
	<li>更改超声波扫描仪上的传感器适配器以匹配探头连接器，然后连接探头。</li>
	<li>运行 Matlab 并激活超声波扫描仪许可证。 注：本节及以下部分含蓄地假设使用威拉索尼奇万塔奇系统。</li>
</ol>3. 超声序列编程
<ol><li>使用示例脚本，设计用于探头定位的传统聚焦"B 模式"（即 echx）序列。<ol>
<li>将成像深度设置为 50 mm。</li>
		<li>将焦距深度设置为 35 mm。</li>
	</ol>
</li>
	<li>使用示例脚本，设计超快超声序列。<ol>
<li>将成像深度设置为 50 mm。</li>
		<li>程序 3 倾斜平面波在 [-3，0，3] 度。</li>
		<li>将脉冲重复频率 （PRF） 设置为 12 kHz。</li>
		<li>使用 4 个半周期进行超声波形式，中心频率取决于所使用的探头。此处假定中心频率为 5.2 MHz。</li>
		<li>将总持续时间设置为 1 s。</li>
	</ol>
</li>
</ol>4. 探头定位和数据采集
<ol><li>在探头的镜头上涂抹超声波凝胶。</li>
	<li>将探针放在幻影上，并启动B模式超声波序列。</li>
	<li>找到感兴趣的运河。液体看起来比周围的组织暗。将探头置于纵向视图中。</li>
	<li>手动将探头保持在感兴趣的位置。</li>
	<li>结束 B 模式序列并启动超快序列获取脚本。</li>
</ol>5. 图像重建 （图 2B）
<ol><li>序列结束后，保存原始数据（也称为射频数据"RF"）。</li>
	<li>使用超声波系统默认软件启动图像重建脚本。在过程结束时，应创建智商数据矩阵。 注：超声波回声记录在探头的每个元素和每个发射/接收，然后存储在 RF 数据矩阵中。图像重建将适当的延迟法应用于每个通道，并导致所谓的"IQ"（阶段/四重）矩阵。复杂的智商矩阵有三个维度：两个用于空间（图像深度和宽度），一个用于时间</li>
</ol>6. 杂乱无章的过滤 （图 2C）
注：有关步骤 6-7，请参阅 补充材料中提供的 Matlab 脚本。
<ol><li>将 3D（空间 x 空间 x 时间）智商矩阵重塑为 2D（空间 x 时间）卡索拉蒂矩阵，名为 IQr。</li>
	<li>计算 IQr 的奇数值分解15 （Eq. 1）。 <img alt="Equation 1" src="/files/ftp_upload/61838/61838equ01v3.jpg"> Eq. 1</li>
	<li>使用巴兰格等人描述的空间单向向量 U计算空间 相似性矩阵 C（II、D），并识别血液亚空间边界 N。</li>
	<li>使用此截止 N 来筛选德梅内等人15 （II，C） 中描述的智商数据。</li>
</ol>7. 流量可视化和速度测量 （图 2C）
<ol><li>通过沿时间维度（Eq.2）集成过滤数据 IQt 的信封来计算功率多普勒地图PD。3D 坐标 z、x 和 t 分别是深度、宽度和时间维度 ，nt 和获得的帧数。 <img alt="Equation 2" src="/files/ftp_upload/61838/61838equ02v5.jpg"> Eq. 2</li>
	<li>以对数比例显示PD地图。要设置动态范围，请计算运河外区域的平均 PD，并将 dB 中的此值用作动态范围的下界值。一个典型的动态范围是[-30，0]dB。</li>
	<li>在图像上定义包含 1 到 30 像素的圆形兴趣区域 （ROI）。</li>
	<li>平均 智商f 信号超过该 ROI 的像素，以获得 <img alt="Equation 3" src="/files/ftp_upload/61838/61838equ03v2.jpg"> nt 时间点的矢量。</li>
	<li><img alt="Equation 4" src="/files/ftp_upload/61838/61838equ04v2.jpg">使用短时间傅立刚变换 （STFT） 的平方级计算和显示多普勒光谱。<ol>
<li>将 STFT 窗口设置为 60 个样本的汉恩窗口。</li>
		<li>将STFT重叠设置为窗口长度的90%。</li>
	</ol>
</li>
	<li>在光谱图的每个时间点叠加中心频率。</li>
	<li>使用多普勒公式（Eq.3）将频率f值转换为血液轴向速度vz。c0是介质中的声音速度，fTW是传输超声波形的中心频率（此处为 5.2 MHz）。 <img alt="Equation 5" src="/files/ftp_upload/61838/61838equ05v4.jpg"> Eq. 3</li>
</ol>

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围绕此协议的主框架，可能会有几种变体。
硬件问题 如果用户提供其自定义主机，主板和计算机的情况必须有一个可用的PCI快递插槽。CPU 还必须有足够的 PCIe 通道来处理所有设备。
探头选择 超声波探头（也称为传感器）是根据所需的空间分辨率和视场的几何形状选择的。探头的中心频率越高，空间分辨率越高，但成像深?...

超声成像是临床实践和研究活动中最常用的成像技术之一。生物组织中超声波发射与后散射回波记录相结合，可以重建解剖图像，即所谓的"B模式"。这种方法完全适用于软组织成像，如生物组织，通常允许超声波穿透几厘米以上，传播速度为≈1540m/s。根据超声波探头的中心频率，可获得分辨率从 30μm 到 1 mm 的图像。此外，众所周知，声源的运动会影响相关波的物理特性。特别是，波相对于其源速的频率变化之间的联系被描述为多普勒效应1，其最简单的表现是移动救护车的警报器的音调变化。超声成像长期以来一直利用这种物理效应来观察移动中的红血球2，并提出了各种通常称为"多普勒成像"的成像模式。这些模式能够评估血液流动在非常不同的应用和器官，如大脑，心脏，肾脏或周围动脉。
值得注意的是，目前大多数可用的超声波系统都依赖于同样的技术，即传统的超声波。基本原理如下：声束使视野发音，并沿着超声波传感器孔径扫射。对于光束的每个位置，回声都会被记录下来并转换成最终图像的行。通过沿传感器逐步移动光束，整个视场可以按线成像（图 1，左面板）。这一战略很好地适应了21世纪初一直存在的电气限制和计算能力。尽管如此，它还是有几个缺点。其中，最终帧速率受光束扫描过程限制为每秒几百张图像。在血流量方面，这种相对较低的帧速率会影响可以检测到的最大流速，这是由香农-奈奎斯特3的采样标准决定的。此外，传统的多普勒必须处理复杂的权衡。为了评估特定感兴趣区域（ROI）的血流量速度，必须连续记录来自该投资回报率的几个回声。这意味着超声波束暂时保持在固定位置。回声合奏的时间越长，对投资回报率的速度估计就越好。但是，要生成视场的完整图像，光束必须扫描介质。因此，人们可以感觉到这两个约束之间的冲突：按住光束精确评估一条线的速度，或者移动光束以生成图像。不同的传统多普勒模式（即彩色多普勒或脉冲波多普勒）直接反映了这种权衡。通常，彩色多普勒会生成用于定位船只4的低保真流图，然后使用脉冲波多普勒准确量化先前标识的容器5中的流量。
这两个限制（低帧率和本地化/量化权衡）都通过非常高帧率的新兴技术来克服。其中，合成孔径方法6或多线传输技术可引证7。在这项研究中，我们专注于所谓的超快超声波方法。二十年前推出的8、9、10，这种方法也依赖于超声波的发射/接收，但模式完全不同。事实上，超高速成像不是使用扫描聚焦光束，而是使用平面波或分散波，这些波能够用单个发射来使视野发出共鸣。在单次发射之后，相关电子产品还能够接收和处理来自整个视野的大量回波。最后，图像可以从单个发射/接收模式11（图 1，右面板）进行重建。由于声能的扩散，这些未聚焦的排放可能具有低信号与噪声比 （SNR）。这可以通过发射几个标题的平面波（或不同来源的发散波）和添加由此产生的图像来解决。这种方法被命名为"连贯复合"12。出现了两个主要后果。首先，帧速率仅取决于飞行的超声波时间，可以达到 1 到 10 kHz 的典型值。其次，这确保了空间和时间维度的数据连续性，也称为空间相干性。因此，传统的本地化/量化权衡被打破。这种高帧速率和空间相干性组合对超声波检测血流的能力有着巨大的影响。与传统的超声波相比，超高速超声波提供了血流3的完整特征。实际上，用户可以访问图像中每个像素的速度时间过程，在整个采集过程中（通常为 ≈1 s），其时间刻度由帧速率给出（通常，帧速率为 5 kHz，时间分辨率为 200μs）。这种高帧速率使该方法适合广泛的应用，如快速流动的器官，如心脏室13或心肌与冠状微注14。此外，它已被证明，其空间的一致性强烈地提高了它的能力，分离缓慢的血液流动从背景移动组织，从而增加了对微血管流15的敏感性。这种能力使16号动物和17号动物都能接触到大脑的微血管。
因此，超高速超声波非常适合在各种情况下的图像血流。它仅限于软生物组织，并将受到硬接口（如骨骼或气腔，如肺）的存在的强烈影响。超声波序列物理参数的调整允许研究减速（下降到1毫米/s 11，16）和快速流动（高达几米/s）。空间分辨率和渗透深度之间存在权衡。通常，分辨率为 50 μm，成本为 5 mm 左右。相反，渗透可以扩展到15-20厘米，成本分辨率为1毫米。值得注意的是，大多数超高速扫描仪，如本文中使用的扫描仪，只提供2D图像。
在这里，我们提出了一个简单的协议，引入超快多普勒成像的概念，使用可编程研究超声波扫描仪和多普勒幻象模仿血管（动脉或静脉）嵌入生物组织。

<table>
	<tbody>
		<tr>
			<td>Blood-mimicking fluid</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>069DTF</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler flow phantom</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>ATS523A</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>MathWorks, Natick, Massachusetts, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump / Doppler flow pump</td>
			<td>CIRS Inc, Norfolk, Virginia, USA</td>
			<td>769</td>
			<td>Include tubings and pulse dampener</td>
		</tr>
		<tr>
			<td>Transducer adpter</td>
			<td>Verasonics, Kirkland, Washington, USA</td>
			<td>UTA 408-GE</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafast ultrasound research scanner</td>
			<td>Verasonics, Kirkland, Washington, USA</td>
			<td>Vantage 256</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound probe/transducer</td>
			<td>GE Healthcare</td>
			<td>GE 9L-D</td>
			<td></td>
		</tr>
	</tbody>
</table>

blood flow imaging with ultrafast doppler

脉冲-多普勒效应是临床血液造影中用于评估血流量的主要技术。适用于传统的聚焦超声多普勒模式，它有几个限制。首先，需要微调信号过滤操作，以区分血液流动与周围移动组织。其次，操作者必须在定位血流或量化血流之间做出选择。在过去的二十年里，超声波成像经历了一个范式的转变，超高速超声波的出现使用了未聚焦的波。除了帧速率增加一百倍（高达 10000 赫兹）外，这项新技术还打破了传统的量化/本地化权衡，为视场提供了完整的血流映射，并同时在单像素级别（降至 50μm）获得精细速度测量。这种空间和时间维度的数据连续性有力地改善了组织/血液过滤过程，从而提高了对小血流量速度的敏感度（降至 1 mm/s）。本文旨在介绍超快多普勒的概念及其主要参数。首先，我们总结了无聚焦波成像的物理原理。然后，我们介绍多普勒信号处理的主要步骤。特别是，我们解释关键组织/血流量分离算法的实际实施，以及从这些过滤数据中提取速度。这种理论描述辅之以体外经验。用流动的血液模拟液嵌入运河的组织幻影与研究可编程超声系统进行成像。获得血流图像，并在运河中显示数个像素的流量特征。最后，建议对体内应用进行审查，在若干器官中举例，如胡萝卜素、肾脏、甲状腺、大脑和心脏。

收购和后期处理的质量首先通过目视检查进行评估。运河的形状必须在功率多普勒图像中清晰可见，组织区域必须显得黑暗。如果多普勒信号的功率不限于运河，则可能意味着杂乱的滤波步骤出错（SVD 阈值太低），或者探测器在获取过程中经历了强烈的移动。
目视检查后，对运河内光谱的研究可以提供实验成败的良好信息。光谱图应是片面的（所有值高于或低于零）。如果?...

Watch this Scientific Journal Video about Blood Flow Imaging with Ultrafast Doppler at JoVE.com

Blood Flow Imaging with Ultrafast Doppler

此协议显示如何应用超高速超声多普勒成像来量化血液流动。经过 1 秒的长收购后，实验者可以访问具有轴向速度值的全视野电影，每个像素每≈0.3 毫秒（取决于飞行的超声波时间）。

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 ...

blood-flow-imaging-with-ultrafast-doppler

Research

Education

JoVE Journal

JoVE Core

Electrical Engineering

Anatomy and Physiology

Bioengineering

Unit 1

Sampling

Blood Vessels and Circulation

SCIENTISTS IN ACTION

7.3K 次观看.  The Hospital for Sick Children, PGCRL Research Institute. 此协议显示如何应用超高速超声多普勒成像来量化血液流动。经过 1 秒的长收购后，实验者可以访问具有轴向速度值的全视野电影，每个像素每≈0.3 毫秒（取决于飞行的超声波时间）。

视频: Blood Flow Imaging with Ultrafast Doppler

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

科研

生物工程

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

微流体流室模型血小板输注与止血措施，实时血小板沉积和纤维蛋白形成

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

使用光学漫反射相关光谱，基于脑血流的休息状态功能连接

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as atomic force microscopy have limited temporal resolution, while F&#246;rster resonance energy transfer (FRET) only allow conformations to be inferred. Fluorescence microscopy, on the other hand, allows real-time in situ visualization of single molecules in various flow conditions. Our protocol describes the steps to capture conformational changes of single biomolecules under different shear flow environments using fluorescence microscopy. The shear flow is created inside microfluidic channels and controlled by a syringe pump. As demonstrations of the method, von Willebrand factor (VWF) and lambda DNA are labeled with biotin and fluorophore and then immobilized on the channel surface. Their conformations are continuously monitored under variable shear flow using total internal reflection (TIRF) and confocal fluorescence microscopy. The reversible unraveling dynamics of VWF are useful for understanding how its function is regulated in human blood, while the conformation of lambda DNA offers insights into the biophysics of macromolecules. The protocol can also be widely applied to study the behavior of polymers, especially biopolymers, in varying flow conditions and to investigate the rheology of complex fluids.

We present a protocol for immobilizing single macromolecules in microfluidic devices and quantifying changes in their conformations under shear flow. This protocol is useful for characterizing the biomechanical and functional properties of biomolecules such as proteins and DNA in a flow environment.

使用荧光显微镜在剪切流下描述单分子构象变化

Single-molecule behavior under mechanical perturbation has been characterized widely to understand many biological processes. However, methods such as ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

高维流式细胞术用于解剖种植体组织的免疫功能分析

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...