AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

在这项研究中动物的工作是经宾夕法尼亚大学的机构动物护理和使用委员会。该方法是按照批准的指导原则进行。 1.准备老鼠和主动脉的分离<ol><li>麻醉用氯胺酮小鼠（80 - 100毫克/千克），赛拉嗪（8 - 10毫克/千克）和乙酰丙嗪（1 - 2毫克/千克）腹膜内。确认尾掐测试麻醉。一旦鼠标被完全麻醉，安乐死通过颈脱位鼠标。 </li><li>将在它的后面鼠标和针鼠标一个夹层板。清洗，用70％的腹部区域（体积/体积）乙醇擦拭。 </li><li>捏住在中线的皮肤，使与腹部中型显微剪一个小切口初期。保持与镊子皮肤，用中等大小的显微剪刀剪开皮肤和腹膜从腹部到胸骨。 </li><li>切去B上的排骨OTH两侧中型显微剪刀。仔细小型显微剪刀取出肺脏和肝脏，并保持心脏和主动脉。鼠标和夹层板传送到解剖显微镜。 </li><li>抓住周围的小镊子主动脉脂肪和用小剪刀小心切掉脂肪主动脉周围。 </li><li>轻轻抓住用镊子主动脉，使一个切口与小型显微剪刀在升主动脉和另一个切口在降主动脉的端部开始，正上方的腹主动脉。 </li><li>转移解剖主动脉到含有1x磷酸盐缓冲的盐水中的60毫米培养皿（PBS;无钙和镁）。 </li><li>继续使用小型显微剪刀主动脉解剖走任何剩余的脂肪组织。用小剪刀纵向打开的主动脉。 </li><li>使用小型显微剪刀剪开降主动脉和aorti的一小片（〜2×4mm的）ç拱原子力显微镜分析（ 图1）。放置在组织中的60毫米的塑料盘和保持水分在PBS一滴。 </li></ol><img alt="图1" src="/files/ftp_upload/54630/54630fig1.jpg" /> 图1:一个图像显示在小鼠的不同主动脉段的位置处的主动脉是从心脏中分离至该膜片，和降主动脉的和一小部分的主动脉弓被用来确定弹性模量。比例尺，1毫米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54630/54630fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.准备组织样本的AFM测量<ol><li>小心使用不接触主动脉实验室擦拭去除PBS。可以肯定，对组织的腔侧朝上。 </li><li>打开主动脉的每一端轻轻粘合到板通过加<html> ING 5 - 10微升氰基丙烯酸酯类粘合剂的用凝胶加载尖（ 图2）。 注:需要在两端牢固安装组织中的胶量必须凭经验确定。至关重要的是，主动脉未在此过程中进行拉伸。 </li><li>检查胶合主动脉一定要确保它没有被折叠或浮动。在主动脉空气干燥该胶后（30 - 60秒），轻轻加入PBS中并浸没样品。 注:制备的原子力显微镜（步骤3 - 5）可以在所述组织被准备用于分析来进行。 </li></ol><img alt="图2" src="/files/ftp_upload/54630/54630fig2.jpg" /> 图2:一个主动脉段的卡通胶合到使用氰基丙烯酸酯粘合剂的60毫米培养皿中的氰基丙烯酸酯粘合剂被施加到主动脉样品的边缘在准备AFM测量。COM /文件/ ftp_upload / 54630 / 54630fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 3.装载探头<ol><li>挂载液体探头支架上探头负载的立场。 </li><li>使用镊子，具有球形前端滑动的氮化硅AFM探针（0.06牛顿/米悬臂）上的探头支架（我们使用了1微米直径的 SiO 2粒子，但也可以使用更大的尺寸）。确保原子力显微镜探针被牢固地固定在探头支架。 </li><li>探头支架滑到Z扫描器安装AFM头，并确保探头支架连接牢固。 </li></ol> 4.校准激光探头上<ol><li>打开AFM软件。选择软件上的实验类型; 实验范畴 ，以接触模式， 实验组 ，联系方式液和实验 ，联系方式液。点击载入EXPeriment在软件;这将开启激光器。 </li><li>加入3毫升蒸馏水至50毫米的玻璃底培养皿并将其放置在激光对准单元的阶段的菜。这个单元简化了激光束聚焦到悬臂的背部的过程。 </li><li>在直立位置上的激光准直单元与功率激光对准单元上安装的AFM头。添加约50微升的蒸馏水，以在针尖的液滴。 </li><li>使用操纵杆，降低AFM头，使针尖使得在菜与水接触。如果触点没有针尖和水盘之间进行，直到接触时轻轻抬起菜。调整对焦，亮度和XY位置，使针尖是鉴于对齐单元的液晶显示屏上。 </li><li>通过调整AFM头的激光定位旋钮激光点到AFM探针的尖端的位置。使用探测器定位旋钮上的原子力显微镜头，调整断定光电二极管直至0和-1 V范围值的离子被用于垂直偏转获得和用于水平偏转得到〜0 V. </li><li>查，以确保该激光和信号被最大化。如果必要，重复步骤4.5至重新调整的针尖的激光束和光电二极管的位置，以获得最大的总和信号的位置。 注意:可能需要重新定位在探头支架上的原子力显微镜探针，以获得即使步骤4.5重复最大和信号。 </li></ol> 5.校准AFM探针的偏转灵敏度和弹簧常数<ol><li>制作与钳子60毫米培养皿划痕和填充用蒸馏水的菜。放置在样品架板培养皿。在这种情况下，安装在倒置显微镜的电动XY扫描阶段的菜。 </li><li>放置在板顶部上的磁性板保持器以保持其在成像期间固定。 </li><li>点击<strong&gt;"上"在移动的AFM头高达近最高位置的软件导航菜单箭头，以确保原子力显微镜探针不会碰的菜。 注意:这一步非常重要，因为它避免断针尖。 </li><li>放置在AFM头显微镜舞台上。 </li><li>使用显微镜把重点放在盘子上的划痕。使用操纵杆，或者单击"向下"箭头在导航菜单中，慢慢地，小心地将原子力显微镜探针进入PBS。在板的刮略高于头位置。 注意:如果AFM头的初始位置更接近样品时，接合时间将显著减少。 </li><li>接合之前，重新调整的针尖的激光束和光电二极管的位置，需要获得最大SUM信号的位置。 </li><li>通过单击更改探头在设置菜单中选择一个提示类型。 </li&gt; <li>点击查看参数和设置参数: 扫描尺寸为0， 扫描速率为1赫兹， 采样/线 256和偏转设定点到20纳米。点击搞。直到探针使得与板的表面接触的锁上状态窗口将保持打开。从事探头后，点击斜坡 。 </li><li>在扫描工具栏菜单中单击扩展模式和设置参数: 斜坡大小为500纳米和1微米的斜率之间，到2赫兹， 样品 256 号 ， 提示半径为500纳米， 样品泊松比为0.5（假设材料待测量是完全不可压缩）， 提示半角为0， 触发模式到相对和触发器阈值 ，以20至100纳米。 </li><li>点击斜坡连续在斜坡工具栏菜单OBTA在盘上（ 图3A）上的力曲线。在这种力曲线，设置通道1 偏差 。 注意:这将允许软件绘制的结果作为垂直偏转与z位置。 <ol><li>点击力曲线的左边或右边结束，拖动光标以包括力曲线（ 图3A，红色虚线线）的线性区域。使用这两条线来标记倾斜区域的边界要适合用一条直线。 </li></ol></li><li>选择斜坡在工具栏，然后单击更新灵敏度 。记录该值并重复此步骤四次。选择在校准工具栏，然后单击探测器 。输入从偏转灵敏度框中五次测量的平均值。 </li><li>点击取款 2 - 3次提高AFM头。这个步骤是重要的，以防止针尖和在热调过程中的板之间的相互作用。单击工具栏热调 。 </li><li>设置1的热调谐范围 - 100千赫（可以从制造商的产品目录中获得该信息）和偏转灵敏度更正为1.144 V形悬臂和1.106矩形悬臂。 </li><li>在热调谐菜单中，单击获取数据以获得热调曲线（ 图3B）。使用AFM软件，代替红色通过从图的边缘在拖动鼠标以限定的边界拟合虚线的曲线（如在图3B示出）的任一侧的行。选择洛伦兹（空），或者根据简谐振荡器（液）的模型上更好地满足数据。 </li><li>点击拟合数据 ，然后计算弹簧K和保存该值。回覆泥炭弹簧常数校准几次，并使用平均值。点击撤消几次。 </li><li>从显微镜舞台中删除AFM头，并把它对准单元。取下显微镜阶段的菜。 </li></ol><img alt="图3" src="/files/ftp_upload/54630/54630fig3.jpg" /> 图3:AFM力曲线用于AFM探针的校准。 （A）代表AFM力曲线（校准曲线）。垂直红之间的作用力曲线的延伸部分虚线来确定悬臂偏转灵敏度。（B）用于计算悬臂如前所述20的弹簧常数的简单谐振子契合图。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54630/54630fig3large.jpg" target="_blank">请点击这里查看一个更大的版本这个数字。</a> 离体 <ol><li>上放置装有显微镜阶段的主动脉组织的60毫米培养皿中，并与磁性板保持固定板。 </li><li>放置在AFM头显微镜舞台上。使一定的AFM探针不与主动脉接触而是轻轻使接触用PBS并形成一个弯月面。如果有必要，将对准单元上的原子力显微镜头和点击撤消直到有足够的空间，以防止接触，然后将AFM头显微镜舞台上。 </li><li>点击导航 。使用操纵杆或点击"下"箭头 ，慢慢降低原子力显微镜探针定位主动脉上方的悬臂。必要时进行调整使用的原子力显微镜头探测器定位旋钮光电二极管的位置。 </li><li>点击锁上以允许悬臂以与主动脉接触。 </li><li>一旦主动脉从事，确保压电中心是稳定的，并单击斜坡 。如果压电中心波动，这可能是一个假啮合的结果。撤回探头和增加垂直的激光的偏移由小增量的光电二极管（〜0.5V）和重新接合。 <ol><li>此外，如果探针是远高于样品表面，手动降低探头接近样品表面之前重新接合。如果前面的步骤不排除波动，尝试更换探头。 </li></ol></li><li>设置斜坡大小为3微米， 触发方式相对和触发阈值至100纳米，然后单击斜坡持续 。观察探针和样品之间的接触点发生在大致中央与Z斜坡周期的下¾。如果这是没有观察到，从样品分离，根据需要进行调整的斜坡尺寸和重新接合样本。 </li><li>在菜单栏中点击显微镜再搞设置 。更改SPM撤 30微米。点击斜坡在斜坡工具栏菜单持续 。 </li><li>观察力曲线后，单击菜单栏中的捕获 ，然后捕获文件名 。与结局".000"（这将允许更多的捕捉文件由1数增加）输入所需的文件名。选择一个指定的文件夹并保存数据。 </li><li>在捕获工具栏菜单中单击捕获 。点击退出 ，然后导航 。使用操纵杆或软件控制在主动脉的另一区域的悬臂定位（旁初始计量点; 见图4）。 </li><li>点击参与 ， 斜坡 ，分别斜坡持续 。观察力曲线后，单击停止 </st荣&gt;，然后捕获 。 </li><li>重复步骤6.9 - 6.10。 如图每区域捕获至少5次测量4 注:我们通常收集15 - 3 25的力曲线-在每个组织5的不同位置， 如图4中分别。 </li></ol><img alt="图4" src="/files/ftp_upload/54630/54630fig4.jpg" /> 图4: -从3 25倍- 5个不同的地点（区域1 - 5）在每个动脉获得整体的刚度悬臂趋近及缩进组织（区域1）的卡通这种AFM重复测量多达15个组织样本。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54630/54630fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 7.数据分析<ol><li>开力曲线分析软件。 </li><li>氯ICK 查找并在菜单栏中打开 ，并加以分析上的文件双击。 </li><li>单击工具栏菜单修改力能参数检查偏转灵敏度，弹簧常数，刀尖半径，提示半角和泊松比。要更改参数，检查它旁边的框，然后在新值列中输入正确的值。然后单击执行 。 </li><li>如果力曲线是嘈杂，单击棚车过滤器 ，并设置为输入方向延伸， 平均分给3和滤波器阶数为0 个 。单击执行平滑数据。 </li><li>点击菜单栏中的基线校正 ，并为方向延伸的输入， 打印单位到部队， 键入要分离， 校正为了 1 日和扩展基准源 </st荣&gt;延伸。调整垂直蓝色虚线线涵盖力曲线的平坦部分，然后单击执行 。 </li><li>在工具栏菜单中单击缩进并设置为活动曲线来扩展， 拟合方法输入点基， 联系点算法来联系治疗如适合的变量， 包括附着力不， 最大力适合边界至30％， 闵力适合边界来0％和拟合模型 ，以赫兹（球形）。 注意:在我们的测量，针尖和样品（在后退曲线悬臂梁的负偏转）之间显著粘附很少观察到。在观察这样的粘合的情况下，适当的模型（DMT或JKR）应该用于分析18,19。 </li><li>减的杨氏模量的值。分析各3的杨氏（弹性）模量-第5区（每区5的力曲线）， 如图4彼此的小的距离内拍摄。 注:要删除的文物，排除杨氏模量&gt; 100千帕（通常〜测量总额的10％）的时间序列。 </li><li>计算每个3的平均杨氏模量的值 - 第5区（已测得），并使用这些值来计算的平均弹性模量为主动脉作为一个整体。 注:至少3，更经常4 - 6个人主动脉被用于获得动脉硬度的准确评估。最后的结果被绘制为"n"的独立实验的平均值±SEM表示。需要主动脉的确切数目将当然取决于精度所要求的水平。 </li></ol>

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	<li>Kothapalli, D., 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=Cardiovascular+Protection+by+ApoE+and+ApoE-HDL+Linked+to+Suppression+of+ECM+Gene+Expression+and+Arterial+Stiffening.">Cardiovascular Protection by ApoE and ApoE-HDL Linked to Suppression of ECM Gene Expression and Arterial Stiffening.</a> Cell Rep. 2, 1259-1271 (2012).</li><li>Liu, S. -. 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=Matrix+metalloproteinase-12+is+an+essential+mediator+of+acute+and+chronic+arterial+stiffening.">Matrix metalloproteinase-12 is an essential mediator of acute and chronic arterial stiffening.</a> Sci Rep. 5, 17189 (2015).</li><li>Lakatta, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+arterial+aging+and+the+epidemic+of+systolic+hypertension+and+atherosclerosis.">Central arterial aging and the epidemic of systolic hypertension and atherosclerosis.</a> J Am Soc Hypertens. 1, 302-340 (2007).</li><li>Stehouwer, C. D. A., Henry, R. M. A., Ferreira, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+in+diabetes+and+the+metabolic+syndrome:+a+pathway+to+cardiovascular+disease.">Arterial stiffness in diabetes and the metabolic syndrome: a pathway to cardiovascular disease.</a> Diabetologia. 51, 527-539 (2008).</li><li>Steppan, J., Barodka, V., Berkowitz, D. E., Nyhan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Stiffness+and+Increased+Pulse+Pressure+in+the+Aging+Cardiovascular+System.">Vascular Stiffness and Increased Pulse Pressure in the Aging Cardiovascular System.</a> Cardiol Res Pract. 2011, 263585 (2011).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Mitchell, G. F., 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=Arterial+Stiffness+and+Cardiovascular+Events:+The+Framingham+Heart+Study.">Arterial Stiffness and Cardiovascular Events: The Framingham Heart Study.</a> Circulation. 121, 505-511 (2010).</li><li>Sutton-Tyrrell, K., 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=Elevated+Aortic+Pulse+Wave+Velocity,+a+Marker+of+Arterial+Stiffness,+Predicts+Cardiovascular+Events+in+Well-Functioning+Older+Adults.">Elevated Aortic Pulse Wave Velocity, a Marker of Arterial Stiffness, Predicts Cardiovascular Events in Well-Functioning Older Adults.</a> Circulation. 111, 3384-3390 (2005).</li><li>Klein, E. A., 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=Cell-Cycle+Control+by+Physiological+Matrix+Elasticity+and+In+Vivo+Tissue+Stiffening.">Cell-Cycle Control by Physiological Matrix Elasticity and In Vivo Tissue Stiffening.</a> Curr Biol. 19, 1511-1518 (2009).</li><li>Bae, Y. H., 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=A+FAK-Cas-Rac-lamellipodin+signaling+module+transduces+extracellular+matrix+stiffness+into+mechanosensitive+cell+cycling.">A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.</a> Sci signal. 7, ra57 (2014).</li><li>Thyberg, J., Hedin, U., Sj&#246;lund, M., Palmberg, L., Bottger, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+differentiated+properties+and+proliferation+of+arterial+smooth+muscle+cells.">Regulation of differentiated properties and proliferation of arterial smooth muscle cells.</a> Arterioscler Thromb Vasc Biol. 10, 966-990 (1990).</li><li>Owens, G. K., Kumar, M. S., Wamhoff, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Regulation+of+Vascular+Smooth+Muscle+Cell+Differentiation+in+Development+and+Disease.">Molecular Regulation of Vascular Smooth Muscle Cell Differentiation in Development and Disease.</a> Physiol Rev. 84, 767-801 (2004).</li><li>Huynh, J., 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=Age-Related+Intimal+Stiffening+Enhances+Endothelial+Permeability+and+Leukocyte+Transmigration.">Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration.</a> Sci Transl Med. 3, 112ra122 (2011).</li><li>Safar, M. E., Levy, B. I., Struijker-Boudier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Perspectives+on+Arterial+Stiffness+and+Pulse+Pressure+in+Hypertension+and+Cardiovascular+Diseases.">Current Perspectives on Arterial Stiffness and Pulse Pressure in Hypertension and Cardiovascular Diseases.</a> Circulation. 107, 2864-2869 (2003).</li><li>Raffetto, J. D., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+and+their+inhibitors+in+vascular+remodeling+and+vascular+disease.">Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease.</a> Biochem Pharmacol. 75, 346-359 (2008).</li><li>Muller, D. J., Dufrene, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscopy+as+a+multifunctional+molecular+toolbox+in+nanobiotechnology.">Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology.</a> Nat Nanotechnol. 3, 261-269 (2008).</li><li>Hsu, B. Y., Bae, Y. H., Mui, K. L., Liu, S. -. L., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E3+Inhibits+Rho+to+Regulate+the+Mechanosensitive+Expression+of+Cox2.">Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2.</a> PLoS ONE. 10, e0128974 (2015).</li><li>Johnson, K. L., Kendall, K., Roberts, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Energy+and+the+Contact+of+Elastic+Solids.">Surface Energy and the Contact of Elastic Solids.</a> Proc R Soc Lond A. 324, 301-313 (1971).</li><li>Derjaguin, B. V., Muller, V. M., Toporov, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+deformations+on+the+adhesion+of+particles.">Effect of contact deformations on the adhesion of particles.</a> J Colloid Interface Sci. 53, 314-326 (1975).</li><li>Hutter, J. L., Bechhoefer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+atomic-force+microscope+tips.">Calibration of atomic-force microscope tips.</a> Rev Sci Instrum. 64, 1868-1873 (1993).</li><li>Pries, A. R., Secomb, T. W., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+surface+layer.">The endothelial surface layer.</a> Pflugers Arch EJP. 440, 653-666 (2000).</li><li>Pogoda, K., 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=Depth-sensing+analysis+of+cytoskeleton+organization+based+on+AFM+data.">Depth-sensing analysis of cytoskeleton organization based on AFM data.</a> Eur Biophys J. 41, 79-87 (2012).</li><li>Mendez, M. G., Restle, D., Janmey, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin+Enhances+Cell+Elastic+Behavior+and+Protects+against+Compressive+Stress.">Vimentin Enhances Cell Elastic Behavior and Protects against Compressive Stress.</a> Biophys J. 107, 314-323 (2014).</li><li>Moreno-Flores, S., Benitez, R., Md Vivanco, ., Toca-Herrera, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+relaxation+and+creep+on+living+cells+with+the+atomic+force+microscope:+a+means+to+calculate+elastic+moduli+and+viscosities+of+cell+components.">Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components.</a> Nanotechnology. 21, 445101 (2010).</li><li>Darling, E. M., Topel, M., Zauscher, S., Vail, T. P., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+human+mesenchymally-derived+stem+cells+and+primary+osteoblasts,+chondrocytes,+and+adipocytes.">Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes.</a> J Biomech. 41, 454-464 (2008).</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> Biophys J. 82, 2798-2810 (2002).</li><li>Mahaffy, R. E., Shih, C. K., MacKintosh, F. C., K&#228;s, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Probe-Based+Frequency-Dependent+Microrheology+of+Polymer+Gels+and+Biological+Cells.">Scanning Probe-Based Frequency-Dependent Microrheology of Polymer Gels and Biological Cells.</a> Phys Rev Lett. 85, 880-883 (2000).</li><li>Amin, M., Le, V. P., Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Testing+of+Mouse+Carotid+Arteries:+from+Newborn+to+Adult.">Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult.</a> J Vis Exp. , e3733 (2012).</li><li>Laurent, S., 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=Expert+consensus+document+on+arterial+stiffness:+methodological+issues+and+clinical+applications.">Expert consensus document on arterial stiffness: methodological issues and clinical applications.</a> Eur Heart J. 27, 2588-2605 (2006).</li><li>Plodinec, M., 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=The+nanomechanical+signature+of+breast+cancer.">The nanomechanical signature of breast cancer.</a> Nat Nano. 7, 757-765 (2012).</li><li>Raman, A., 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=Mapping+nanomechanical+properties+of+live+cells+using+multi-harmonic+atomic+force+microscopy.">Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy.</a> Nat Nano. 6, 809-814 (2011).</li></ol>

The authors have nothing to disclose.

AFM压痕可用于表征细胞和组织的刚度（弹性模量）。在本文中，我们提供详细的一步一步的协议来隔离小鼠降主动脉和主动脉弓，并确定这些地区的动脉离体的弹性模量。我们现在总结和讨论在本文所描述的方法的技术问题和限制。 几个技术问题可以在隔离，并给予他们的小而薄的自然鼠标主动脉的分析产生。当清洗过的动脉被纵向打开，必须小心不要破坏那里的弹?...

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table><tbody><tr><td>BioScope Catalyst AFM system</td><td>Bruker</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE 200 inverted microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Silicon nitride AFM probe</td><td>Novascan Technologies</td><td>PT.SI02.SN.1</td><td>0.06 N/m cantilever; 1 &amp;micro;m SiO&lt;sub&gt;2&lt;/sub&gt; particle</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>See section 1.4</td></tr><tr><td>Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>See section 1.8</td></tr><tr><td>Fine Scissors-ToughCut</td><td>Fine Science Tools</td><td>14058-11</td><td>See section 1.4 (medium sized)</td></tr><tr><td>Vannas-T&amp;uuml;bingen spring scissors</td><td>Fine Science Tools</td><td>15008-08</td><td>See section 1.6 (small sized)</td></tr><tr><td>60 mm TC-treated cell culture dish</td><td>Corning</td><td>353004</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1x</td><td>Corning</td><td>21-031-CM</td><td>Without calcium and magnesium</td></tr><tr><td>Krazy Glue instant all purpose liquid</td><td>Krazy Glue</td><td>KG58548R</td><td>See section 2.2</td></tr><tr><td>Gel-loading tips, 1 - 200 &amp;micro;l</td><td>Fisher</td><td>02-707-139</td><td>See section 2.2</td></tr><tr><td>Tip Tweezers</td><td>Electron Microscopy Sciences</td><td>78092-CP</td><td>See section 3.2</td></tr><tr><td>50-mm, clear wall glass bottom dishes</td><td>TED PELLA</td><td>14027-20</td><td>See section 4.4</td></tr></tbody></table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

动脉硬化是一个显著风险因子和生物标志物用于心血管病和老化的标志。原子力显微镜（AFM）是用于多种材料从硬（塑料，玻璃，金属等 ）表面的任何基底上细胞的表征粘弹性的机械性能的多功能的分析工具。它已被广泛地用于测量细胞的刚度，但不经常用于测量主动脉的刚度。在本文中，我们将介绍使用AFM在接触模式下测量空载小鼠动脉离体弹性模量的过程。我们描述程序的鼠标主动脉隔离，然后提供的AFM分析的详细信息。这包括用于激光束的对准，弹簧常数和原子力显微镜探针的偏转灵敏度的校正，并采集力曲线的一步一步的说明。我们还提供了数据ANALY了详细的方案力曲线的妹妹。

<html> 图5A示出了从6个月大的，雄性C57BL / 6小鼠的降（胸）主动脉的相位对比图像。原子力显微镜悬臂是直接发生在组织上面并准备缩进。 图5B和5C展示在接触模式下的AFM压痕获得代表力曲线。在图5B和5C示出绿线表示使用球体赫兹模型中获得的最佳拟合曲线。在图5D中 ，如在文中所描述的降主动脉和主动脉弓的平均刚度从...

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

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 ...

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

10.4K 次观看.  University of Pennsylvania. We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

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

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

科研

生物学

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, ...

生物工程

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

测量升主动脉刚度<em&gt;在体内</em&gt;小鼠使用超声

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

医学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

机械性刺激，集成光学和原子力显微镜研究活细胞的反应

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

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 ...

小鼠视网膜毛细血管和内皮下基质硬度的测量

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

作者聚焦：研究早期糖尿病视网膜病变的血管僵硬和炎症

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.

测量的刚度

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