作者感谢保罗Janmey博士在宾夕法尼亚大学提供本文中使用的细胞株。 QW JF拜菲尔德和埃文安德森也承认他们独到的讨论AFM技术。

1。校准的弹簧常数悬臂<ol><li>根据制造商的指示，加载到AFM的悬臂。任何实验之前，有必要清洁的悬臂支架用乙醇。这将有助于限制在AFM测量细菌污染文化。 </li><li>校准InvOLS的（逆光杠杆感光度）。此参数描述光电二极管的响应（伏特）每纳米悬臂偏转量。 </li><li>到样品台上装入干净的载玻片上，然后安装在原子力显微镜头和调整激光束和对应根据制造商的指示。搞针尖于载玻片上。 </li><li>与压电撤回，重新调整镜子的光电二极管读-2 V.执行力光谱测量触发点（最大光电二极管的响应）+2 V。 </li><li>与压电撤回，重新调整镜子的光电二极管读-2五执行FORCE光谱测量触发点（最大光电二极管的响应）+2 V 注：这里的电压值是具体 ​​到庇护AFM。此值应根据制造商所提供的规格设置。 数据采集​​完成后，公司接触区域的力曲线放大。对这个地区进行的线性拟合，求出斜率，这将是在V / nm处。该值的倒数描述的悬臂光电二极管集成的光灵敏度。 </li><li> 0复位镜对准一个自由偏转V。 </li><li>校准的悬臂弹簧常数。甲热调整方法是用来确定在悬臂28的弹簧常数。 </li><li>校准InvOLS后，提起扫描仪从样品台不存在任何探针和样品之间的相互作用。 </li><li>开始捕捉热数据。在此过程中，悬臂梁的热振动是原始记录四。原子力显微镜的软件分析这种热振动，并将其绘制在一个数据窗口中的功率谱。 </li><li>几秒钟后，数据采集，在最低的频率（基波谐振）的峰确定的弹簧常数为中心的数据段进行拟合。 </li></ol> 2。装样<ol><li>在AFM阶段安装盘加热器配件，如果不是已经配备。 </li><li>将温度设定到37℃，并等待系统达到一个稳定的热平衡20分钟。 </li><li>放置在培养皿上的原子力显微镜的阶段，然后将它固定采用与烹调加热器提供钳位。重要的是要除去从孵化器中的盘子，并把它在舞台上，以免损伤细胞之间的时间最小化。长于30分钟测量时，CO 2的独立的介质，应使用以取代正常的培养基。 </li><li>应用37℃培养基t的冰山一小滴他AFM悬臂，并降低原子力显微镜头，直到提示刚刚浸没在液体中。 </li><li>使用顶视图，CCD相机，重新调整的悬臂（在液体中比在空气中会有所不同的取向，由于介质的折射率的变化）上的激光束。 </li><li>搞针尖在一个干净的培养皿面积。 </li><li>执行校准InvOLS，如上所述，在液体环境中的悬臂梁的灵敏度。 </li></ol> 注：a）如培养细胞水凝胶，校准InvOLS应进行预先对充满了细胞培养基的培养皿的底表面。当切换到细胞样本，特别注意要支付不改变激光束对准的悬臂。二）InvOLS重新校准，每当有一个变化中的激光准直。三）被还建议采取InvOLS，平均价值从几个校准曲线，S因斯每次校准产生不同的InvOLS的。 InvOLS的变化，但是，小的比的平均值。例如，校准布鲁克DNP-10悬臂弹簧常数0.06 N / m的液体的100倍，产生一个的平均InvOLS价值66.3纳米/ V标准偏差仅为0.5纳米/ V。 3。收集细胞压痕力曲线<ol><li>选择一个单元格缩进。借助光学显微镜，移动载物台的位置，使尖端位于在细胞核周围区域的单元格上方的悬臂。可以通过应用的X和Y扫描仪的偏移量的精确调节的悬臂位置。 注：AFM悬臂移动样品阶段从样品表面，而选择靶细胞被撤回。保护悬臂从敲到样品，由于样品表面可能并不平坦。 </li><li>切换到强制光谱模式。设置缩进率提高到10微米/秒，低到足以避免流体动力效应的范围内。 </li><li>设置偏转触发点，从而限制了最大缩进力，以避免对细胞的伤害。选择相对触发选项，这将纠正任何在偏转信号漂移。 2 NN的最大力量是一个很好的起点，对于大多数样品。然而，此值，应调整根据示例刚度。对于软样品，应使用一个较低的值，以避免过度压痕样品。对于僵硬的样品，应使用高值来产生可测量压痕。 </li><li>设置足够大，以确保的前端将被完全脱离的细胞之间的力的测量的力的距离。一般，力的距离被设定在5微米。 </li><li>命令的AFM采取单一的力曲线。 </li><li>收集至少三力曲线，在不同的地点，在原子核周围地区的每一个细胞中。虽然这是有利于采取多可靠的统计数据，在每个单元上的曲线，过多服用力曲线，可导致细胞刚度的变化，由于应力的AFM探针。 </li><li>当数据收集完成后，撤回的提示，并重复步骤3.1-3.6尽可能多的细胞，需要一定的样本条件下对细胞刚度良好的统计数据。通常情况下，30个细胞为每个条件测定。 </li></ol>刚度单个细胞内的分布特征，力地图模式。在警队地图模式，设置扫描尺寸，包括该地区的利益，设置一个合适的分辨率设置缩进参数选择单力曲线，AFM然后将光栅跨越定义的样本区域，并采取单一的力量曲线在样品区中的每个像素。 4。数据分析力曲线记录使用一个自定义的MATLAB程序来计算单元刚度分析。以下是MATLAB程序的简要说明： <ol><li> MATLAB程序标识的接触点的坐标Z0和D0（ 见图1），使用的算法从公布的方法，通过林等29： </li><li>力曲线中的每一个数据点，兴趣点的左侧的执行的数据的线性拟合，赫兹模型适合的权利（使用所选择的点作为初始的接触点），到设定的最大压痕（建议200-300纳米）。 </li><li>对于每一个点，计算相对误差两者的拟合和总结这些值。 </li><li>的总拟合误差达到最小的点被选择为初始联系点。 注：可以减少计算时间，通过实施一个黄金分割的搜索，而不是线性扫描整个部队曲线。 </li><li>试样变形δ和缩进力F的计算公式为： <img alt="公式1"FO：内容宽度=“3.073in”FO：SRC =“/ files/ftp_upload/50497/50497eq1highres.jpg”SRC =“/ files/ftp_upload/50497/50497eq1.jpg”/&gt; </li><li>使用最小二乘拟合，以适应F与δ在后接触区的数据和z≥Z 0，赫兹模型中提取的杨氏模量，E的细胞： <img alt="公式（2）" fo:content-width="2.773in" fo:src="/files/ftp_upload/50497/50497eq2highres.jpg" src="/files/ftp_upload/50497/50497eq2.jpg" /> ， 其中，v为泊松比。 注意：当δ是10％以上的样品的厚度（单元格的高度），所测得的细胞刚度受基板的刚度。核周边区域的厚度通常为几微米的量级。因此，只有第一个200-300纳米的F-δ曲线适合的赫兹的模型。 </li></ol>

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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=Absence+of+filamin+A+prevents+cells+from+responding+to+stiffness+gradients+on+gels+coated+with+collagen+but+not+fibronectin.">Absence of filamin A prevents cells from responding to stiffness gradients on gels coated with collagen but not fibronectin.</a> Biophysical Journal. 96, 5095-5102 (2009).</li><li>Cross, S. 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=AFM-based+analysis+of+human+metastatic+cancer+cells.">AFM-based analysis of human metastatic cancer cells.</a> Nanotechnology. 19, 384003 (2008).</li><li>Storm, C., Pastore, J. J., MacKintosh, F. C., Lubensky, T. C., 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=Nonlinear+elasticity+in+biological+gels.">Nonlinear elasticity in biological gels.</a> Nature. 435, 191-194 (2005).</li><li>Wen, Q., 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=Polymer+physics+of+the+cytoskeleton.">Polymer physics of the cytoskeleton.</a> Current Opinion in Solid State &amp; Materials Science. 15, 177-182 (2011).</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 (02), 2798-2810 (2002).</li><li>Trache, A., Lim, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+response+to+mechanical+stimulation+studied+by+integrated+optical+and+atomic+force+microscopy.">Live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy.</a> J. Vis. Exp. (44), e2072 (2010).</li><li>Lim, S. M., Kreipe, B. A., Trzeciakowski, J., Dangott, L., Trache, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+effect+on+RhoA+signaling+modulation+in+vascular+smooth+muscle+cells.">Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells.</a> Exp. Cell Res. 316, 2833-2848 (2010).</li></ol>

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AFM的压痕法表征力学性能的活细胞的优点。尽管不那么敏感的磁扭仪和光学镊子，它可以测量的力量piconewton 32级，原子力显微镜能检测阻力纳米牛顿微微牛顿从几十到几百不等，从样品比较力范围可以应用到使用微量吸管19的细胞。此范围内的力适合需要建立可测量的变形，在所有类型的细胞19。高空间分辨率，使得它可以组织中，于单电池33上的亚微米级特性的...

的机械性能，特别是刚性，单个细胞和其周围的细胞外基质（ECM）的许多生物过程，包括细胞生长，运动，分裂，分化，和组织的平衡是至关重要的。1已经证明，主要是由细胞的机械刚度细胞骨架，特别是肌动蛋白和中间纤维和与它们相关联的其他蛋白质的网络2上在体外的网络的肌动蛋白和中间丝的机械性能试验的结果表明，细胞力学很大程度上依赖于细胞骨架结构和预加应力的细胞骨架3-5刚度的活细胞，然后把作为指标来评价细胞骨架结构6，肌球蛋白活性7和许多其他细胞的过程。更重要的是，在细胞的机械特性的变化也经常发现有密切associated与各类疾病，如肿瘤的形成和转移。8-10监控的机械刚度活细胞，因此可以提供一种新的方式来监控细胞生理学检测和诊断疾病，以及药物治疗的有效性进行评估。 12 已经开发了多个方法，包括粒子跟踪微流变学，13-16磁捻流式细胞仪，17微吸管18,19和20-22显微测量细胞的弹性。粒子跟踪微流变学的痕迹注入细胞或细胞内的细胞骨架的基准标记或亚微米的荧光颗粒的热振动23细胞的弹性和粘性性质从测量粒子位移涨落耗散定理计算14,23此方法允许同时测量的地方高空间分辨率的机械性能在不同的地方，在一个单元格。然而，注入到细胞中的荧光发色粒子，可能会导致细胞功能的变化，细胞骨架结构，因此细胞力学。微管吸吮方法适用于微量的直径范围从1到5μm至细胞膜的一小片进入移液管吸负压。细胞刚度的计算是由所施加的负压力和细胞膜的变形18此方法，但是，不能检测刚度整个单元中的非均匀分布。磁性捻流式细胞仪（MTC）施加磁场，超顺磁性珠连接到细胞膜上产生转矩。来自在该方法中的（17）细胞的刚度，从所施加的转矩之间的关系和细胞膜的扭转变形。这是很难控制的位置磁珠在MTC方法，并且它是也是challengi的ng来表征高分辨率的扭曲变形。显微适用于压头具有良好定义几何冲进入细胞。在细胞的缩进力以及由此产生的压痕往往遵循赫兹模型的预测。的杨氏模量，可以计算出从细胞的力压痕曲线拟合赫兹模型。这种方法已被广泛地应用到测试的组织和细胞的机械性能，尽管它的局限性，如在接触点的确定，赫兹模型的适用性，以及潜在的物理损伤，使细胞的不确定性。在众多的设备在microindentaion 20，原子力显微镜（AFM）是市售的，并已被广泛应用来表征力学性能的活细胞和组织中21,24-27。 本文演示了使用一个的庇护MFP3D生物AFM表征细胞力学过程。 AFM不上光年提供高分辨率的地形细胞也已被广泛应用到组织细胞的机械性质。 AFM压痕的原则，在图1中示出。原子力显微镜悬臂梁接近从几微米以上的细胞与细胞接触;缩进的单元格，这样的悬臂偏转达到预选的设定点;和拉相差的细胞。在此过程中被记录，作为其位置的函数，如在图1中所示的悬臂偏转。在与细胞接触之前，悬臂移动介质中，没有任何明显的偏转。缩进时对细胞，悬臂弯曲和偏转信号的增加。被建模为悬臂弹性梁，以使它们的偏转是在该室施加的力成比例。通过设置最大悬臂挠度，施加在样品上的力的最大量值被限制到避免d豪悦国际细胞。的力的部分，从点b，点c在图1中 ，进入细胞的前端缩进，适合赫兹模型，提取细胞的刚度曲线。 <img src="/files/ftp_upload/50497/50497fig1.jpg" alt="图1" fo:content-width="4.75in" fo:src="/files/ftp_upload/50497/50497fig1highres.jpg"/> 图1。 AFM显微插图和解释力曲线顶部面板显示驱动压电扫描器AFM悬臂的运动。悬臂式z和悬臂偏转信号 d的垂直位置被记录在这个过程中。从点a，几微米的单元格上方的悬臂。在接近该单元格，样品压痕δ保持为零，直到它到达b点的前端开始与细胞接触。 b点的坐标中的情节是临界值对数据进行分析，记为（Z 0，D 0&gt;）。从b到c的悬臂的悬臂偏转进入细胞内的缩进，直到达到设定点时，它被设置为对象的最大的缩进力和悬臂弹簧常数之间的比值。 ，一旦偏转信号达到预先设定的最大值，然后撤回悬臂从点d，它经常被向下拉由于小费样本粘附细胞，从细胞中分离并返回到其初始位置在e。右边的窗格中示出的缩进和记录的z和D信号之间的关系。在左下面板是一块有代表性的力曲线，以悬臂的最大压痕，其中的弹簧常数测定为0.07N /米，被设定为17纳米，以便最大缩进力施加到样品是1.2 NN。在压痕标记的关键位置。

<table border="1">
<tbody>
<tr>
<td>Atomic Force Microscope </td>
<td>Asylum Research</td>
<td>MFP3D-BIO</td>
</tr>
</tbody>
</table>

measuring the mechanical properties of living cells using atomic force microscopy

细胞及细胞外基质（ECM）的机械性能，在许多生物过程中扮演重要的角色，包括干细胞的分化，肿瘤的形成，和伤口愈合。细胞和ECM刚度的变化往往是在组织细胞的生理或疾病变化的迹象。因此，单元的刚度是一个指数来评估细胞培养的状态。在人群中的用于测量细胞和组织的刚度的方法，使用原子力显微镜（AFM）的显微压痕提供了一种方法来可靠地测量活细胞的刚度。这种方法已被广泛地应用于表征微观尺度刚度，为各种材料，包括金属表面的生物软组织和细胞。这种方法的基本原理是，缩进一个细胞与一个选定的几何形状的针尖，并测量所施加的力从原子力显​​微镜悬臂梁弯曲。力压痕曲线拟合赫兹模式l为相应的尖端几何能给材料刚度的定量测量。本文演示程序，利用原子力显微镜的活细胞的刚度特性。关键步骤包括的AFM校准的过程中，力曲线采集，数据分析使用MATLAB例程证明。这种方法的局限性进行了讨论。

图2a显示了三个有代表性的塑料表面，聚丙烯酰胺凝胶的杨氏模量每年3,000 17,000霸，分别取自3T3成纤维细胞培养力曲线。经过仔细吞吐量曲线中的接触点，作为细胞变形的功能的缩进力被绘制在图2b中。根据级小于0.3 nN的，金字塔形尖缩进3微米，为3千帕的聚丙烯酰胺凝胶上培养的细胞的一个力。与此相反，需要的力超过1.6 nN的缩进使用相同的尖端，在普通培养皿的细胞?...

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Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

本文演示了一个协议，通过使用原子力显微镜（AFM）的显微表征活细胞的机械性能。

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, ...

measuring-mechanical-properties-living-cells-using-atomic-force

Research

JoVE Journal

Bioengineering

39.6K 次观看.  Worcester Polytechnic Institute. 本文演示了一个协议，通过使用原子力显微镜（AFM）的显微表征活细胞的机械性能。

视频: Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

科研

生物工程

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

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 ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

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

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 the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

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 ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces.

细菌表面电位测量用开尔文探针力显微镜

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

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 ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

利用合成生物学工程师活细胞接口，具有可编程材料

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

荧光技术结合测定活细胞中的力敏感蛋白动力学

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...