Name: fabrication and implementation of a reference-free traction force microscopy platform
Uploaded: 2019-10-06T14:30:00
Description: Watch this Scientific Journal Video about Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform at JoVE.com

O. A. Banda得到了NSF IGERT SBE2研究金（1144726）、特拉华大学提供的启动基金以及国家卫生研究院/国家癌症研究所IMAT项目（R21CA214299）的资助。JHS得到国家卫生研究院/国家癌症研究所IMAT计划（R21CA214299）和国家科学基金会CAREER奖励计划（1751797）的资助。显微镜访问由NIH-NIGMS（P20 GM103446）、NSF（IIA-1301765）和特拉华州提供资助。结构化照明显微镜是使用特拉华州联邦研究与发展赠款计划（16A00471）获得资金购买的。用于双光子激光扫描光刻的LSM880共焦显微镜是使用共享仪器授予（S10 OD016361）获得的。

1. PEGDA基水凝胶的光聚合
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收集试剂
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<li>收集锂苯基-2，4，6-三甲基苯甲酸酯（LAP），3.4 kDa聚（乙酰）乙二醇二甘醇（PEGDA），n-乙烯基丙酮（NVP），AlexaFluor 488标签PEGDA（PEG-488），AlexaFluor 633标记PEGDA（PEG-633），和PEGylatedPEG-RGDS）从各自的冰柜中，使每个冰柜都室温。</li>
		<li>在单独的琥珀微离心管中，测量3毫克LAP、10毫克PEGDA、5毫克PEG-488、20mgPEG-633和6mgRGDS肽。</li>
	</ol>
</li...

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该协议的目标是提供一个工作流，以缓解与生成和分析 TFM 数据相关的许多困难。一旦制备，光图案水凝胶易于使用，只需要了解标准组织培养实践和荧光显微镜。无参考方面允许在充满细胞的氢凝胶上无忧无虑地导航，并消除繁琐的图像处理步骤，如参考图像和变形图像之间的图像配准。生成的分析几乎完全自动化，从各个 COA 的数据可以从头到尾分析不到 10 分钟。
该协议...

牵引力显微镜 （TFM） 是使用附着细胞和收缩细胞生成的基准标记的插值位移场近似细胞牵引力的过程。使用TFM，可以研究细胞外环境中的机械线索对重要的细胞过程的影响，如增殖、分化和迁移。，5，6，7，8，9，10，11，12。 遗憾的是，许多现有方法可能难以实现，或者需要熟悉高度专业化的分析和计算工具，从而使没有经验的研究人员难以使用 TFM。我们描述了一种生成 TFM 平台的方法，该方法消除了分析中的一些困难，同时还提供高吞吐量数据采集。
在现有的TFM方法中，最常用的用于量化材料应变的方法包括将小荧光标记物（通常是纳米或微米大小的荧光珠）加入可变形的水凝胶中，如聚丙烯酰胺（PAA）或聚烯酰胺（PAA）或聚烯酰胺（乙二醇）二甲苯甲酸酯（PEGDA）13，14，15 。这些基于珠的方法提供了在感兴趣的细胞周围密集聚类基准标记的能力，以最大化位移采样。不幸的是，磁珠在整个水凝胶中的分布不能直接控制，因此空间组织是随机的。这种随机放置会导致诸如珠子太近而无法准确解决等问题，或者扩散到基板的斑块产生低质量的数据。无法预测基准标记在没有单元格的情况下的位置也会产生一个约束，对于每组收集的单元牵引数据，还必须捕获处于宽松状态的基础标记的附加参考图像。需要参考图像，以便可以近似于强调图像和无应力图像之间的差异。为了达到放松状态，被测量的细胞要么化学放松，要么完全去除。此过程通常阻止进一步的实验测量，抑制长期细胞研究，并限制吞吐量。参考图像还需要图像配准技术来适应实验期间可能发生的漂移，这通常导致将应力状态图像与参考图像进行繁琐的手动匹配。
其他被视为无参考的TFM方法，通过高分辨率光刻、微接触印刷或微成型16、17、18对基准标记的分布实施某种形式的控制，19，20.无参考 TFM 是通过假设每个基准标记的宽松状态可以根据制造过程中标记位置的指定方式预测的。这些方法允许在单个图像捕获中完全捕获细胞的张力状态，其中基准标记位移与隐含参考相比，比从基准标记几何中推断的要低。虽然标记放置的一致性通常使用这些平台来实现，但相对于广泛使用的基于珠的方法，它们通常有其自身的缺点，包括：1） 牵引力降低;2） 降低平面外位移的精度（在某些情况下完全无法测量）;3） 平台基材和材料的可定制性降低（例如配体表示、机械性能）。
为了解决这些缺点，我们设计了一个新的无参考TFM平台。该平台利用多光子活性化学将少量荧光荧光酸与水凝胶内的特定 3D 位置交联，作为测量材料应变的基准标记。通过这种方式，我们设计了一个平台，其操作方式与基于珠子的方法类似，但具有显著优势的是，基准标记被组织成网格数组，允许无参考材料应变跟踪。这种无参考属性提供了许多优点。首先，它允许对细胞牵引状态进行非侵入性监测（即，避免需要放松或移除细胞以获得置换基准标记的参考位置）。这是我们设计该系统的主要目标，因为我们打算将其他下游分析方法与 TFM 结合使用，这与破坏性的端点 TFM 方法可能很困难。其次，使用基于网格数组的隐含引用可实现几乎完全自动化的位移分析。数组的规律性创建了一个可预测的工作流，其中异常情况（即包含意外伪影（如次优标记间距或注册不匹配）的样本单元格数据的发生可以保持在最低限度。第三，无需获取参考图像，即可在较长时间内对单个样本上的多个单元进行监视。这与传统的基于珠的方法形成鲜明对比，根据显微镜自动级运动的保真度，定位误差会累积并增加正确记录参考图像以控制细胞张力的难度。图像。总体而言，该平台有助于提高收集蜂窝张力数据的吞吐量。
通过该协议，我们希望让读者熟悉我们实现的双光子激光扫描光刻技术，以生成这种无参考的 TFM 平台，以测量由种子细胞生成的平面内和平面外牵引组件表面上。该协议未涵盖的一些单体组分的合成。一般来说，这些反应包括几乎相同的"一锅"合成反应方案，前面描述的21，和这些产品的替代品也可以购买。我们还致力于让读者熟悉我们生成的基于软件的工具，以推广使用市售的激光扫描显微镜作为 3D 打印工具，并促进基准标记位移的分析。

<table>
	<tbody>
		<tr>
			<td>Acrodisc Syringe Filter, 0.2 &#956;m Supor Membrane, Low Protein Binding</td>
			<td>Pall</td>
			<td>PN 4602</td>
			<td>Allows for filtering of macromer solutions prior to base gel synthesis and subsequent lithography steps.</td>
		</tr>
		<tr>
			<td>Acrylate-Silane Functionalized #1.5 Coverslips</td>
			<td>in-house</td>
			<td>in-house</td>
			<td>Acrylates allow binding of base hydrogel to the glass surface to immobilize the hydrogels. See reference: 21-24</td>
		</tr>
		<tr>
			<td>Axio-Observer Z1 w/Apotome</td>
			<td>Zeiss</td>
			<td></td>
			<td>Widefield microscope with structured illumination module used to capture images for TFM.</td>
		</tr>
		<tr>
			<td>Chameleon Vision ii</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td>Equipped on laser-scanning microscope used for multiphoton Lithography.</td>
		</tr>
		<tr>
			<td>Double Coated Tape, 9500PC, 6.0 mil</td>
			<td>3M</td>
			<td></td>
			<td>Binds acrylate-silane functionalized coverslips to Petri dishes.</td>
		</tr>
		<tr>
			<td>Flexmark90 PFW Liner</td>
			<td>FLEXcon</td>
			<td>FLX000620</td>
			<td>Allows lining of double coated tape enabling feeding of tape into plotter.</td>
		</tr>
		<tr>
			<td>LSM-880</td>
			<td>Zeiss</td>
			<td></td>
			<td>Laser-Scanning microscope used for Multiphoton Lithography.</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2018a</td>
			<td>Runs custom scripts to generate lithography instructions for microscope and for analysis of TFM data.</td>
		</tr>
		<tr>
			<td>Model SC Plotter</td>
			<td>USCutter</td>
			<td>SC631E</td>
			<td>Cuts double coated tape into rings to bind coverslips to petri dishes.</td>
		</tr>
		<tr>
			<td>Objective C-Apochromat 40x/1.20 W Corr M27</td>
			<td>Zeiss</td>
			<td></td>
			<td>Equipped on both widefield microscope and laser-scanning microscope to be used for both lithography and TFM.</td>
		</tr>
		<tr>
			<td>PEG-AF633</td>
			<td>in-house</td>
			<td>in-house</td>
			<td>Fluorophore-labeled acrylate PEG variant for creating fiducial markers. See reference: 21</td>
		</tr>
		<tr>
			<td>PEG-DA</td>
			<td>in-house</td>
			<td>in-house</td>
			<td>Base material for hydrogels. See reference: 21</td>
		</tr>
		<tr>
			<td>PEG-RGDS</td>
			<td>in-house</td>
			<td>in-house</td>
			<td>RGDS peptide-labeled mono-acrylate PEG variant for promoting cell-adhesion. See reference: 21</td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>CELLTREAT</td>
			<td>229638</td>
			<td>8mm holes are cut into the center of each dish using a coring bit to fit base hydrogels.</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>For creating spacers to control base hydrogel thickness (aka PDMS).</td>
		</tr>
		<tr>
			<td>Syringe, Leur-Lok, 1 mL</td>
			<td>BD</td>
			<td>309628</td>
			<td>Allows for filtering of macromer solutions prior to base gel synthesis and subsequent lithography steps.</td>
		</tr>
		<tr>
			<td>UV Lamp</td>
			<td>UVP</td>
			<td>Blak-Ray&#174; B-100AP</td>
			<td>Polymerizes base hydrogel.</td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409-5G</td>
			<td>Radical accelerant and co-monomer. Improves pegylated fluorophore incorporation during lithography.</td>
		</tr>
	</tbody>
</table>

fabrication and implementation of a reference-free traction force microscopy platform

量化细胞引起的材料变形提供了有关细胞如何感知和响应其微环境的物理特性的有用信息。虽然有许多方法可用于测量细胞引起的材料应变，但这里我们提供了一种以无参考方式以亚微米分辨率监测应变的方法。使用双光子激活光刻图案化过程，我们演示如何生成机械和生物主动可调合成基板，其中包含嵌入式荧光基准标记阵列，以轻松测量三维 （3D） 材料变形轮廓，以响应表面牵引力。使用这些基板，可以使用感兴趣的细胞的单个 3D 图像堆栈映射细胞张力曲线。我们采用这种方法的目标是使牵引力显微镜成为研究细胞干扰转导过程的研究人员，尤其是该领域新手更容易获得和更容易实施的工具。

在整个协议中，有许多检查点提供反馈，以评估模式化过程的质量。为了提供有关如何评估每个检查点的进度的见解，我们提供实际实验的代表性结果。研究结果强调了该协议在用于人类脐带内皮细胞（HUVECs）的TFM分析制备的光型水凝胶上的应用。结果包括原始图像数据以及每个关键步骤的处理数据输出。
第一个检查点发生在步骤 4，一旦基准标记阵列在水凝胶中进行了光图...

Watch this Scientific Journal Video about Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform at JoVE.com

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

该协议提供了用于实施多光子光刻的指令，以制造嵌入聚（乙二醇）水凝胶中的荧光基准标记的三维阵列，用作无参考、牵引力显微镜平台。使用这些说明，简化了 3D 材料应变的测量和蜂窝牵引力的计算，以促进高通量牵引力测量。

无参考牵引力显微镜平台的制造和实施

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

牵引力显微镜用于测量细胞产生的力。该协议简化了牵引力数据的收集分析，以便让没有经验的用户更容易访问。与现有的无参考牵引力显微镜平台不同，该技术的主要优点是多光度光刻光刻能够根据单个实验需求进行快速、简单的图案设置重新设计。在构建和实施这个新的无参考牵引平台时，有许多关键步骤，这些步骤通过可视化表示而不是仅通过文本更容易理解。对于基础水凝胶的光聚化，首先将三微升的预聚合物溶液添加到薄薄的、扁平的全氟脱氧气碱板上，并将平坦的 150 微米厚 PDMS 条放在周围，但与聚合物前溶液不接触。在 PDMS 上放置丙烯酸硅化盖玻片，预聚合物液滴居在盖玻片下，将预聚合物液滴压到 PDMS 垫片的厚度。将夹层预聚合物溶液暴露在紫外线下约一分钟。当水凝胶完全形成时，小心地将盖玻片与 PDMS 分隔器分离。使用高性能双面丙烯酸粘合剂将打开的底部培养皿连接到盖玻片上。将充满水凝胶的盖玻片粘附在培养皿上时，请特别小心。湿盘或应用过程中压力过小，会漏油，破坏样品。向粘合剂接触面施加压力，在盖玻片、粘合剂和培养皿之间形成完整的密封，注意不要打碎玻璃。使用无菌过滤的 PBS 冲洗水凝胶。然后，在为水凝胶合成准备的实验室溶液的 800 微升中添加 8 微升 NVP。要将二进制图像转换为数字掩码，请打开 MATLAB 并打开并运行 runscript。m MATLAB 脚本。选择要转换的二进制 TIF 文件，然后选择一个文件夹来保存区域的文件。输入所选图像所需的最终大小（以微米）。输入图像将缩放和标注尺寸以匹配这些参数。若要创建单个像素数组，请在"感兴趣区域生成器选项"中，取消选中"小区域/单个像素"下的"删除 tic"框，选中"正方形"框，取消选中"水平断线下使用"。然后，单击"确定"。OVL 文件将在以前指定的文件夹中找到。在显微镜软件中打开文件，在两次光子激光扫描光刻过程中加载控制激光快门所需的区域。应在光线极小的条件下执行以下步骤。在低光照条件下制造基准标记阵列，将先前准备的 NVP 实验室解决方案的 200 微升与 20 毫克 Alexa Fluor 633 彻底混合，并从含有水凝胶的培养皿中去除所有 PBS。将PEG-633混合物加入水凝胶，作为完全包含基水凝胶的液滴，将Peri盘加载到显微镜阶段的样品支架上。然后盖上盘子，让图案溶液浸泡在水凝胶中至少30分钟，不受光线的影响。要配置显微镜进行成像，请选择适当的激光器和滤镜来可视化 Alexa Fluor 488。使用 PEG-488 信号定位水凝胶，并阻止从探测器收集更长的发射波长。使用垂直和水平切片扫描定位 XY 平面中的水凝胶中心，并在此位置将舞台归零。使用 Z 堆栈函数中的线扫描来定位水凝胶的表面，并在此位置将焦点归零。通过重复从 XY 中心之外的线扫描来识别表面位置，根据需要调整显微镜阶段的固定螺钉，对水凝胶进行调平。在显微镜软件工作区中，创建一个单独的光模型实验文件，将多光子功率设置为 1.8%，扫描速度设置为 6。调整图像帧和像素的大小，以实现像素大小为 0.1 微米/像素，纵横比为 100 比 1，然后将区域文件加载到区域选项卡中。使用宏设置所有区域以获取和打开 Z 堆栈函数。将间距设置为 3.5 微米，总深度为 28 微米，Z 切片总数为 9 个。然后使用"位置"函数在水凝胶上设置特定位置，其中将采用光模式进行基准标记阵列。当浸泡时间接近 30 分钟标记时，每五分钟获取一次水凝胶表面的 Z 堆栈线扫描，以根据表面相对于零对焦位置的位置变化检查膨胀。如果在五分钟间隔内表面位置未发生更改，请运行阵列设置并使用 488 纳米激光器验证水凝胶表面在阵列过程中未移动。然后从显微镜上取出水凝胶，从培养皿中吸出PEG-633溶液，然后用无菌过滤的PBS冲洗。要获取细胞和基准标记图像，请将培养皿返回样品架以定位图案区域。找到感兴趣的细胞并获取该细胞的传输或荧光图像。然后获取单元格下方图案数组的 Z 堆栈（如演示），并获取第二组细胞的传输或荧光图像。收集图像堆栈时，生成的图像应显示一个常规的图案特征数组，这些特征的强度会随着图像堆栈中 Z 位置的函数而振荡。追踪m 脚本提供了多个诊断图像，包括用于评估预处理质量的荧光模型标记的 Z 投影、检测到的质心图、作为 Z 位置的函数用于评估对象检测质量的颜色，以及表示已检测到的标记中心的轨道图，这些轨迹已链接到 Z 方向的列中，以评估对象跟踪质量。DISP 3D.m 脚本提供强度图，作为给定图像堆栈中每个检测到要素列的 Z 位置的函数，以评估基准标记强度配置文件的质量。两个 DISP 3D。m 和 DISP 剪切。m 一起提供每个笛卡尔坐标尺寸中测量的位移噪声的直方图以及位移的插值热图。此外，中间final3D_2。m 提供使用外包代码计算的表面牵引力的热图。在此过程过程中，要记住的最重要的事情是，包含 LAP 的解决方案对光敏感，应尽可能保护环境光源。请记住，NVP 是一种挥发性有机化合物，应始终在化学流罩中处理。

Research

JoVE Journal

Bioengineering

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

科研

生物工程

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

干膜 Photoresist-based 电化学微流控生物传感器平台: 器件制备、片上检测制备及系统运行

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-ÃƒÅ½Ã‚Â² on the Epithelial-to-Mesenchymal Transition

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

实现干扰反射显微镜,实现无标签、高速微管成像

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...