Name: stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope
Uploaded: 2018-07-03T14:00:00.000+00:00
Duration: 7 min 2 s
Description: Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

我们感谢玛格丽特卡洛斯·加德尔慷慨地允许使用流变仪。我们承认 NIH (1R15GM116082) 支持这项工作。

1. 软硅基板的制备
<ol><li>使用 (聚苯乙烯) 称重托盘, 从软硅胶弹性体套件中称量1.75 克的 a 组分和1.75 克 (a: b = 1:1)。</li>
	<li>将一个组件添加到称量托盘中的 B 组件中, 并用适当的涂抹棒将其混合在一起5分钟。</li>
	<li>将上述混合物添加到35毫米培养皿中。允许混合物均匀地分布在培养皿上几分钟。 注: 培养皿直径的选择和软硅胶的用量将决定软硅的厚度。在这里, 厚度将在3.5 毫米左右;有关在讨论部分中选择弹性体厚度的更多信息。</li>
	<li>将培养皿与硅胶混合物, 用盖子关闭, 在真空室中15分钟, 以消除任何气泡。在这段时间内, 预热热板到70摄氏度。</li>
	<li>一旦热板达到70摄氏度, 放置一个玻璃幻灯片, 然后将培养皿与硅胶混合物在玻璃滑梯上。让硅胶在70摄氏度治疗30分钟。不要将聚苯乙烯盘子直接放在热板上, 因为培养皿可能会融化。</li>
</ol>2. 荧光微珠与软硅胶的耦合
<ol><li>将固化的软硅胶 (在未发现的培养皿中) 放在深紫外腔内 (带有深紫外灯波长为185和 254 nm 的外壳)。将软硅胶样品 (离紫外线灯约 5-10 厘米) 暴露到深紫外光中5分钟。<ol>
<li>当硅胶接触到深紫外光时, 请继续下面的步骤 2.2-2.6。深紫外照射后, 在检索样品前加加深紫外线室至少5分钟。</li>
	</ol>
</li>
	<li>同时, 在1.5 毫升离心管中称量出19毫克 1-乙基-3-(3-dimethylaminopropyl) carbodiimide (EDC), 并向其添加 500 ul 的去离子水 (DI) 水。轻轻摇动管子即可溶解。</li>
	<li>在一个单独的1.5 毫升离心管, 体重11毫克的n-hydroxysulfosuccinimide (磺 NHS), 增加 500 ul 的 DI 水, 并通过轻轻摇动管溶解磺 NHS。然后, 将磺和 NHS 的解决方案结合在一个离心管中。</li>
	<li>对于此磺-NHS 解决方案, 添加 30 ul 0.44 微米直径红色 (或任何其他荧光颜色基于过滤器立方体可用的荧光显微镜) 羧酸改性荧光微珠 (1% 瓦特/v 库存浓度)。</li>
	<li>对磺-NHS/珠混合, 添加0.02 毫克的胶原蛋白 I (从鼠尾, 库存浓度4毫克/毫升在0.02 米乙酸), 以获得约0.02 毫克/毫升浓度。</li>
	<li>漩涡的 EDC/NHS/珠/胶原蛋白 I 混合物简要地确保珠均匀分散在整个, 前联轴器。</li>
	<li>吸管1毫升的 EDC/NHS/珠/胶原蛋白 I 混合在一块 parafilm 放在另一个浅, 扁平盖 (直径较小) 上。在这种混合物上反转培养皿与软硅胶, 使软硅表面接触的混合物, 但不直接触及较小的培养皿盖子表面下面。要提高倒置培养皿, 使用一个或两个玻璃幻灯片在任何一侧的倒置培养皿间隔。 注: 请参阅图 1以了解如何执行步骤2.7。</li>
	<li>用铝箔盖住样品, 室温下孵化30分钟。</li>
	<li>用软硅胶去除培养皿, 并将其直立 (硅胶侧向上)。</li>
	<li>用磷酸盐缓冲盐水 (pbs) 将2毫升的 PBS (7.4) 加入到盘子中, 以此来洗涤柔软的硅胶表面。让它坐几分钟。用2毫升的 pbs 将 pbs 吸掉, 再用硅胶清洗。让硅胶治疗大约一天。因此, 将软硅胶样品放在 PBS 中过夜37摄氏度。</li>
</ol>3. 用 Widefield 荧光显微镜测量球形压痕的硅硬度
<ol><li>使用软硅胶检索培养皿, 并确保它包含至少1毫升的 PBS, 使硅表面在液体表面以下几毫米。</li>
	<li>使用尖镊子, 在软硅胶上滴下五1毫米锆球形购买。将球体浸入液体介质中, 除去它们, 远离硅胶层的边缘, 至少 5 indentor 直径远离其他购买的位置。 注: 在液体表面上跌落时, 由于液体介质的表面张力, 球体可能无法进入液体介质 (浮子)。</li>
	<li>将培养皿与软硅胶放在显微镜台上, 使其能够通过培养皿的底座进行成像。</li>
	<li>使用10X 目标的相位成像 (如0.30 的干10X 目标), 定位并将球体 indentor 为焦点。</li>
	<li>采取一个部分或整个 indentor 的相位图像, 并保存该图像。如果可用, 请使用平铺扫描。如果 indentor 有任何可见的缺陷, 则丢弃并将其替换为另一个 indentor。</li>
	<li>在实时相位成像下, 平移到 indentor 边缘的左侧, 使框架的左边缘至少距 indentor 中心 1.5 R 的距离。确保 indentor 的中心在右侧保持可见, 靠近图像框架的右边缘。采取相图并保存它。</li>
	<li>将显微镜光源切换为红色荧光通道的光照。与x和y坐标不变 (indentor 中心的xy位置在框架的右边缘附近), 焦点向下 (减少 Z) 直到红色荧光微珠在球形 indentor 的中心之下别再专注了</li>
	<li>对于每 z 增量为0.5 µm 的 z 堆栈, 取一个 z-栈, 直到硅树脂顶层的微珠从 indentor (靠近成像框架的左边缘) 离开焦点。</li>
	<li>对示例中的其他购买重复步骤 3.4-3.8。</li>
</ol>4. 计算硅胶的刚度 (杨氏模量)
<ol><li>使用 ImageJ 打开 indentor 的相位图像, 单击直线工具, 并测量 indentor 的直径 (以像素为单位)。单击并按住 indentor 边缘上的某个点, 将光标移动到边缘上的截然相反的点上, 并记下在释放游标之前在 ImageJ 主窗口的状态栏上显示的像素长度。<ol>
<li>通过单击 "分析", 确保长度单位设置为像素: 设置刻度并检查长度单位。</li>
		<li>考虑到目标放大倍数和 ccd 相机像素尺寸 (r 在微米 = r 像素 x, ccd 相机像素大小为微米/目标放大倍数), 将 indentor 的半径以像素转换为微米。</li>
	</ol>
</li>
	<li>打开红色通道 z 栈的 microbead 图像 (如果微珠是红色荧光) 在 ImageJ 通过点击文件 |进口 |图像序列并选择堆栈中的任何图像, 然后单击"确定" 打开堆栈。 注: F1 是 indentor 中心下的微珠最可能聚焦的帧数, 而 F2 是在框架的左边缘附近的微珠 (位于离珠中心 1.5 R 的区域) 的帧数, 在最可能的焦点。两帧之间的z差是压痕深度δ。<ol>
<li>使用 ImageJ 中的 "线条" 工具, 在图像中的定义良好的 microbead 之间绘制一条线。点击分析 |绘制剖面图并单击 "实时" 按钮, 在选择不同的帧时, 通过珠子获得更新的线扫描强度。提供最大强度值的帧可以选择为焦点的帧。</li>
		<li>由于 z 堆栈中的帧之间的 z 增量为0.5 微米, 因此计算在微米中的缩进深度为δ = (F2-F1) x 0.5。</li>
	</ol>
</li>
	<li>计算在凝胶上施加的力由 indentor 由于它的重量 (减去反对的浮力), 即压痕力 F, 作为 indentor x 的容量 (indentor 的密度-液体媒介的密度) x 加速度由于重力。 使用等式F = (4/3) x 3.142 x (R3) x (ρindentor -ρ媒介) x g那里R是 indentor 的半径, ρindentor是密度 indentor, ρ媒介是液体介质和g的密度是由于重力引起的加速度 (9.81 米/秒2)。用 SI 单位的右手边表示所有数量, 以获得F (N)。</li>
	<li>使用修改后的11赫兹模型12方程计算硅树脂的杨氏模量 (E): <img alt="Equation 1" src="/files/ftp_upload/57797/57797eq1.jpg"> 地方 c = 修改其后面的赫兹模型表达式的修正因子; v = 泊松比的硅胶凝胶 (采取0.5 作为不可压缩材料7); F = 压痕力; R = indentor 半径;和 δ = 压痕深度。 在宾夕法尼亚州的右侧用 SI 单位表示所有数量以获得E<ol>
<li>计算校正因子c如下3: <img alt="Equation 2" src="/files/ftp_upload/57797/57797eq2.jpg"> 地方 <img alt="Equation 3" src="/files/ftp_upload/57797/57797eq3.jpg"> <img alt="Equation 4" src="/files/ftp_upload/57797/57797eq4.jpg">;和 <img alt="Equation 5" src="/files/ftp_upload/57797/57797eq5.jpg">. 应该注意的是, 这个校正因子是专门用于只有当软硅胶坚持良好的培养皿 (或硬硅树脂) 下 (这是这里的情况)。</li>
		<li>根据加硅量和培养皿直径计算软硅层的高度 h。或者, 直接获得 h 通过确定硅层的顶部和底部表面的 z 坐标由相位成像 (次要杂质进入焦点在任一表面)。注意, 对于大 h (h2 > Rδ), 修正因子c接近1。</li>
	</ol>
</li>
	<li>对每个 indentor 重复步骤 4.1-4.4。平均从每个 indentor 获得的杨氏模量, 以获得有机硅样品的平均杨氏模量。</li>
</ol>

<ol>
	<li>Handorf, A. M., Zhou, Y., Halanski, M. A., Li, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stiffness+dictates+development,+homeostasis,+and+disease+progression.">Tissue stiffness dictates development, homeostasis, and disease progression.</a> Organogenesis. 11 (1), 1-15 (2015).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proceedings of the National Academy of Sciences. 94 (25), 13661-13665 (1997).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79, 144-152 (2000).</li><li>Discher, D. E., Janmey, P., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+Substrate.">Tissue cells feel and respond to the stiffness of their Substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in Cell Biology. 83, 29-46 (2007).</li><li>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li><li>Style, R. W., 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=Traction+force+microscopy+in+physics+and+biology.">Traction force microscopy in physics and biology.</a> Soft Matter. 10 (23), 4047-4055 (2014).</li><li>Lee, E., 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=Deletion+of+the+cytoplasmic+domain+of+N-cadherin+reduces,+but+does+not+eliminate,+traction+force-transmission.">Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.</a> Biochemical and Biophysical Research Communications. 478 (4), 1640-1646 (2016).</li><li>Frey, M. T., Engler, A., Discher, D. E., Lee, J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+methods+for+measuring+the+elasticity+of+gel+substrates+for+cell+culture:+microspheres,+microindenters,+and+atomic+force+microscopy.">Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy.</a> Methods Cell Biol. 83, 47-65 (2007).</li><li>Lee, D., Rahman, M. M., Zhou, Y., Ryu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement.">Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement.</a> Langmuir. 31 (35), 9684-9693 (2015).</li><li>Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+elastic+moduli+of+thin+layers+of+soft+material+using+the+atomic+force+microscope.">Determination of elastic moduli of thin layers of soft material using the atomic force microscope.</a> Biophysical Journal. 82 (5), 2798-2810 (2002).</li><li>Hertz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Beru&#776;hrung+fester+elastischer+Ko&#776;rper.">&#220;ber die Beru&#776;hrung fester elastischer Ko&#776;rper.</a> Journal f&#252;r die reine und angewandte Mathematik. 92, 156-171 (1882).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M., Cassimeris, L., Tran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+a+direct+printing+protocol+using+deep+UVs.">Protein micropatterns: a direct printing protocol using deep UVs.</a> Microtubules: In Vivo. , 133-146 (2010).</li><li>Bashirzadeh, Y., Qian, S., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-intrusive+measurement+of+wall+shear+stress+in+flow+channels.">Non-intrusive measurement of wall shear stress in flow channels.</a> Sensors and Actuators A: Physical. 271, 118-123 (2018).</li><li>Muhamed, I., Chowdhury, F., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+tools+to+study+cellular+mechanotransduction.">Biophysical tools to study cellular mechanotransduction.</a> Bioengineering (Basel). 4 (1), 12 (2017).</li><li>Dumbali, S. P., Mei, L., Qian, S., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+sheet-averaged+tension+within+a+large+epithelial+cell+colony.">Endogenous sheet-averaged tension within a large epithelial cell colony.</a> Journal of Biomechanical Engineering. 139 (10), 101008 (2017).</li></ol>

作者没有什么可透露的。

球形压痕法易于实现, 但要注意 indentor 的选择和软硅试样的厚度。用于计算杨氏模量的方程式在一组条件下有效11, 当硅胶样品的厚度 > 10% 的 indentor 半径和 < ~ 13 x indentor 半径时, 通常会满足这些要求。我们发现, 硅树脂厚度为 5-10x 的 indentor 半径是一个很好的选择, 其中试样厚度不太高 (即目标工作距离不成为限制), 计算刚度也不太敏感的硅胶厚度的精确值。球形 indentor 的选择也应该是这样, 压痕深度δ是 < 10% 的硅胶厚度以及 < 10% 的 indentor 半径。考虑到这些因素, 不同材质和直径的购买可以用来测量柔软和坚硬的有机硅的刚度。压痕深度的确定是该协议最关键的步骤。本协议中提出的识别聚焦图像的方法应有助于可靠地确定压痕深度。还应注意的是, 球面压痕法的刚度计算采用赫兹理论, 假定无摩擦接触。在这里, 这是一个很好的假设购买低粗糙度。虽然我们使用了特定的软硅弹性体 (列出在附表中的特定试剂), 其他商用硅胶弹性体套件可以使用。请注意, 广泛用于微细加工的硬硅树脂不是在人民军范围内制造刚性基底的好选择。然而, 柔软的有机硅 (在人民军射程的下端有刚度) 可以与少量的硬硅树脂混合, 使基板在人民军射程的更高端有刚度。根据弹性体, 可以选择不同大小或密度的 indentor, 只要前面提到的条件满足。
荧光微珠与软硅树脂的顶面耦合的几个关键因素是很重要的。首先, 我们选择了0.44 µm 羧酸珠, 因为它们的荧光含量, 因此亮度比较小的类似珠。如果珠子含有较亮的显影, 则可以使用较小的珠子尺寸, 但我们建议使用亚微米羧酸珠, 以免对该方法的分辨率产生不利影响。硅胶表面与 EDC/sulfo-NHS/bead/col1 混合物的孵化是在一个倒置的配置中与硅胶表面进行的。其原因是, 当与珠子的混合物放在硅表面的顶端, 珠块沉淀到硅胶表面, 导致一个糟糕的空间分辨率, 而荧光微珠正在拍摄。即使有了这个协议, 偶尔也会观察到珠子团簇 (图 3中顶部图像中的明亮区域)。但是, 它们不够广泛, 无法影响方法的分辨率。也可以使用图 1C中任何一个培养皿的边缘下的间隔, 使硅酮表面接触液体, 而不是它下面的固体表面。如果选择了含疏水性涂层的微球, 即使没有深紫外光步, 微珠与软硅胶顶面的耦合也可以进行。刚度测量是在最终基板 (UV 处理后, 珠耦合, ECM 耦合) 上进行的, 在哪些单元可以被镀。应牢记, 在采取步骤 (如 UV 处理) 后, 应执行刚度特性, 从而可能改变基板刚度, 从而使测量的刚度成为单元格所能接触到的刚性。
而不是培养皿, 一板硬的可用作软硅胶14的基础。这种配置可用于将外部拉伸应用到细胞中, 其中刚性硅胶提供了可以拉伸的框架, 而软硅胶提供了一种更具生理性的细胞微环境。牵引力显微镜15,16也可以执行与镀在这些软硅胶凝胶7,8, 和荧光微珠的存在, 只有在顶层, 使良好的分辨率与只是一个 widefield 荧光显微镜。胶原蛋白 i 在这个协议可以被替换与其他细胞外基质蛋白。与原子力显微镜类似的方法相比, 球面压痕法可以更容易地实现。使用球面压痕法获得的平均杨氏模量与使用流变仪确定的偏差通常 < 10%10。因此, 球形压痕法 (使用 widefield 荧光显微镜) 为美国匹茨堡中应用的软硅 (或水凝胶) 刚度的定量提供了一种可访问的方法。

软组织细胞位于微环境中, 其刚度在 kilopascal 范围为1, 与组织培养皿的硬度为几级以上。早期的细胞外基质蛋白涂层软基板的实验表明, 基底刚度影响细胞的移动以及坚持在2,3下的细胞外基质。事实上, 基底刚度从根本上影响细胞功能4 , 其方式类似于弥漫的生化信号。聚丙烯酰胺凝胶 (涂有细胞外基质蛋白) 是 (水渗透) 凝胶, 已广泛用作细胞培养基质的美国匹茨堡研究5。烷是最常见的有机硅 (聚硅氧烷), 广泛应用于兆帕斯卡硬度为微米级制造6的硬硅树脂。最近, 在生理上相关的 kilopascal 范围内有刚性的软硅基板被使用作为细胞培养基质为美国匹茨堡研究7,8。
采用了几种方法测量柔性基板的刚度, 包括原子力显微镜、拉伸、流变和压痕时全试样的宏观变形, 球面和球尖 microindentors 9..虽然每种技术都有自己的优缺点, 但用球体压痕是一种特别简单而又相当精确的方法, 只需要进入 widefield 荧光显微镜。在前工作3、9、10中, 用金属球体压痕测量水凝胶的刚度。早期的工作表明基底刚度对细胞运动的重要性, 利用这种方法来确定水凝胶基板刚度3。最近, 共焦显微术也被用于一个优雅的特征10。
在这里, 我们提出了一个逐步的协议, 以准备一个软的硅基板, 耦合荧光珠 (和细胞外基质蛋白, 如胶原 i) 只是到顶端表面, 成像一个缩进球和顶面使用阶段和荧光成像, 分别对图像进行分析, 计算出硅基板杨氏模量。这种方式制备的软硅基板可以方便地用于牵引力显微实验。使用硬硅树脂 (而不是培养皿) 作为软硅胶的基础也使美国匹茨堡研究使用外部拉伸。如有必要, 还指出了避免可能出现的并发症所需的实际考虑。

<table><tbody><tr><td>CY 52-276 A/B silicone elastomer kit</td><td>&amp;nbsp;Dow Corning</td><td>CY 52-276</td><td>Store at room temperature</td></tr><tr><td>Thermo Scientific Pierce EDC</td><td>Fisher Scientific</td><td>PI22980</td><td>Store at -20&amp;deg;C</td></tr><tr><td>Thermo Scientific&amp;nbsp;Pierce Sulfo-NHS crosslinker</td><td>Fisher Scientific</td><td>PI-24510</td><td>Store at 4&amp;deg;C</td></tr><tr><td>Carboxyl fluorescent pink particles, 0.4-0.6 &amp;micro;m, 2 mL</td><td>Spherotech, Inc.</td><td>CFP-0558-2</td><td>Store at 4&amp;deg;C, do not freeze</td></tr><tr><td>1.0 mm Acid washed Zirconium beads</td><td>OPS Diagnostics LLC</td><td>BAWZ 1000-250-33</td><td></td></tr><tr><td>Deep UV chamber with ozone evacuator</td><td>Novascan Technologies, Inc.</td><td>PSD-UV4, OES-1000D</td><td></td></tr><tr><td>Wide field fluorescence microscope</td><td>Leica Microsystems</td><td>DMi8</td><td></td></tr><tr><td>Collagen I, from rat tail</td><td>Corning</td><td>354236</td><td>Stock concentration = 4 mg/ml; store at 4&amp;deg;C</td></tr><tr><td>ImageJ-NIH</td><td>N/A</td><td>N/A</td><td>public-domain software</td></tr></tbody></table>

stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope

人体软组织通常在 kilopascal (kPa) 范围内有刚度。因此, 硅胶和水凝胶柔性基质已被证明是有益的基质, 培养细胞在物理微环境中, 部分模拟体内条件。在这里, 我们提出了一个简单的协议, 以表征杨氏模量的各向同性线性弹性基板通常用于美国匹茨堡研究。该协议包括在培养皿或硬硅树脂上制备软硅基板, 用荧光珠涂覆硅基板的顶面, 使用毫米级球体缩进顶面 (按重力), 成像荧光用荧光显微镜对缩进硅胶表面上的珠子进行分析, 并对所合成的图像进行解析, 计算出硅基板的杨氏模量。将基体的顶面与模数胞外基质蛋白耦合 (除了荧光珠), 使硅基板易于用于细胞电镀和随后的研究使用牵引力显微镜实验。使用硬质硅胶, 而不是培养皿, 作为软硅胶的基础, 使使用美国匹茨堡研究涉及外部拉伸。该协议的一个具体优点是, widefield 荧光显微镜在许多实验室中是常见的, 是本程序所需的主要设备。我们通过测量不同弹性模量的软硅基板的杨氏模量来证明该协议。

使用上面详细的协议, 我们准备了软硅胶在一个35毫米培养皿, 治愈它在70°c 30 分钟和耦合的荧光微球 (和胶原 i) 到顶端表面, 如图 1所示。深紫外线已用于最终的蛋白质耦合到基板13。请注意, (I) 这里使用的固化条件是特定于这种软硅胶和 (II) 压痕测量是在第二天进行, 因为软硅胶有望在一天的过程中进一步治疗。
图 2A显示了硅酮表面球形压痕的各种参数。相位成像用于捕获 indentor 的整个图像 (I), 如图 2B所示 (如果需要, 使用图像拼接) 或 (II) 球体图像的一部分。从 indentor 的图像中提取的唯一参数是其直径。例如, 对于在本协议中使用1:1 软硅胶的 indentor, 来自同一批次的不同个体购买的直径从950µm 到1200µm, 平均值为1037µm, 标准偏差为47µm (8 购买)。请注意, 为特定的 indentor (而不是平均直径为许多购买) 测量的直径应用于压痕的刚度计算-由该特定的 indentor 引起。
硅胶顶面上的微珠的荧光图像是在xy框架位置拍摄的, 因此 indentor 下的区域位于框架的最右侧部分。框架左上部分的区域被选择为远离 indentor 的区域, 如图 3所示。图 3也显示了 indentor 下的区域和远离 indentor 的 Z 堆栈图像。对于使用1:1 软硅胶的1毫米直径锆 indentor, 2 个区域聚焦的 z 值与大约20µm (δ) 不同。这比软硅胶的厚度小得多, 大约有3500µm. 使用 indentor 锆 (实际上是由二氧化锆和二氧化硅混合物制成) 的密度 (4.66 克/厘米3) 和液体的密度中等 (为 PBS: 1.01 克/cm3), 在硅树脂施加的净力量可以被计算。在考虑的情况下, 它是在 20-25 µN 范围内。我们为1:1 软硅胶计算的杨氏模量是 7.2 2.4 帕 (从28个位置汇集从6独立样品)。代表的结果为其他 A: B 比率为同样软的硅胶 (在伴生的具体试剂表中指定) 在表 1中给出。最后, 为了验证使用 widefield 显微镜的球形压痕方法, 我们在本协议中所描述的, 我们还测量了一个聚丙烯酰胺凝胶的杨氏模量, 我们的特点是流变仪有一个年轻的模数为 21 3 帕。利用本协议的球形压痕方法, 使用 widefield 显微镜, 发现同一成分的聚丙烯酰胺凝胶具有 22.1 4.2 帕的杨氏模量, 表明了良好的协议10。在讨论部分中讨论了在执行这些测量时应注意的问题。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57797/57797fig1v2.jpg"> 图 1: 将荧光微珠与软硅胶顶面耦合的过程示意图.(A) 已固化的软硅胶暴露在深紫外光下5分钟. (B) 在水中, 磺、pipetted、珠子和胶原蛋白的混合物, 被放在一个直径较小的盖子上的 parafilm 上。(C) 软硅胶样品倒置在这种混合物, 使它是与液体接触, 但不与顶部表面的较小盖子下方。两个玻璃幻灯片两侧, 培养皿下, 作为间隔。(D) 用 PBS 清洗样品后, 用荧光微珠涂覆的软硅表面可以进行刚度测量。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57797/57797fig1v2large.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/57797/57797fig2.jpg"> 图 2: 软硅胶表面球形压痕的示意图.(A) 这种示意图描述显示了一个软硅胶样品表面的球形 indentor。指出了各种感兴趣的参数。(B) 这个小组显示了一个图像, 1 毫米 indentor (在软硅胶样品) 获得了通过相位成像。刻度条指示250µm.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57797/57797fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57797/57797fig3.jpg"> 图 3: 珠形图像的采集和聚焦图像的确定.(A) 此荧光图像显示了软硅胶样品的顶面上的微球, 以及相对于 indentor (虚线) 所需的框架的xy位置。刻度条指示150µm. 面板B和C显示了软硅胶表面 (B) 下 indentor 和 (C) 以外区域的 z 堆栈荧光图像 (顶部图像中的盒装区域)。z1 和 z2 的指标分别对应于 indentor 和该区域远离 indentor 的区域的 z 值。刻度条指示20µm。单色图像显示的是那些在红色通道, 因为名义上的粉红色微珠是使用的激发和发射剖面适合红色通道。(D) 这个面板显示了一个微珠的强度线扫描 (显示在嵌入图像中的黄色线横跨), 因为焦点是不同的 z 增量0.5 µm。与聚焦图像对应的焦点 (z 值) 可以根据与最大强度的线扫描对应的 z 值进行客观选择。插入图中的刻度条表示20µm.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57797/57797fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本</a>。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>硅胶弹性体 * A: B</td>
			<td>杨氏模量 ** (帕)</td>
		</tr>
<tr>
<td>1:1</td>
			<td>7.2±2。4</td>
		</tr>
<tr>
<td>4:7</td>
			<td>37.6±3。9</td>
		</tr>
<tr>
<td>1:2</td>
			<td>64.1±6。9</td>
		</tr>
<tr>
<td colspan="2">* 在附表中指明的特定试剂/设备 ** 用球面压痕法测量, 使用 widefield 显微镜作为本协议的详细说明</td>
		</tr>
</tbody></table>表1。杨氏模量的软硅胶 (针对特定的硅胶表中指定的具体试剂/设备) 的不同成分的测量使用此处详述的协议.两个混合组件的比率值 A: B (和相应的测量数量) 为 1:1 (28)、4:7 (13) 和 1:2 (8)。

Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

kilopascal 范围内的刚性基底对于研究细胞对生理相关的微环境刚度的反应是有益的。使用一个 widefield 荧光显微镜, 年轻的软硅胶凝胶的弹性模量可以确定使用一个合适的球压痕。

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

stiffness-measurement-soft-silicone-substrates-for-mechanobiology

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

6.8K 次观看.  Old Dominion University. kilopascal 范围内的刚性基底对于研究细胞对生理相关的微环境刚度的反应是有益的。使用一个 widefield 荧光显微镜, 年轻的软硅胶凝胶的弹性模量可以确定使用一个合适的球压痕。

视频: Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

基质贴壁细胞的微管吸吮的估计单元刚度

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

科研

生物工程

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

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, ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

发育生物学

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

单轴拉伸应变下活细胞核动力学的高分辨率成像

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

生物学

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

使用原子力显微镜结合偏振显微镜测量肝硬度

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

摘要

探索更多视频

此视频中的章节

基质贴壁细胞的微管吸吮的估计单元刚度

使用原子力显微镜测量活细胞的力学性能

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

在机械拉伸活细胞成像

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

单轴拉伸应变下活细胞核动力学的高分辨率成像

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

数百个 C.白化病细胞生物原子力显微镜测量自动化

测量的刚度

使用原子力显微镜结合偏振显微镜测量肝硬度

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

摘要

探索更多视频

此视频中的章节

基质贴壁细胞的微管吸吮的估计单元刚度

使用原子力显微镜测量活细胞的力学性能

简单的聚丙烯酰胺为基础的多井刚度检测方法的刚度依赖的细胞反应的研究

在机械拉伸活细胞成像

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

单轴拉伸应变下活细胞核动力学的高分辨率成像

生物物理检测探头相间细胞核的力学性能：基材应变的应用和微针操纵

数百个 C.白化病 细胞生物原子力显微镜测量自动化

测量的刚度

使用原子力显微镜结合偏振显微镜测量肝硬度

数百个 C.白化病细胞生物原子力显微镜测量自动化