笔者想感谢埃琳娜Lomakina，理查德·Bauserman，玛格丽特杨曼，吉Vaknin，杰西卡·斯奈德，克里斯Striemer，纳库尔Nataraj，红栗橱嗯的，TEJAS Khire，和Eric林对他们的援助，这个项目的。该项目是由美国国立卫生研究院PO1 HL 018208。

1。方法 1.1细胞流动室甲流室， 如图1中所示，构建了使细胞可为1.0帕（10达因/厘米2）的剪切下生长，然后直接转移到一个的庇护MFP3D原子力显微镜（圣巴巴拉，加利福尼亚州）。 <ol><li>流动室，制备实验首先通过清洗载玻片的Piranha溶液（3:1 H 2 SO 4：H 2 O 2）为15分钟，然后用蒸馏水洗涤。然后将它们进行烘烤，以干燥和涂有氨基丙基三乙氧基硅烷（APTES）在真空沉积室中。 </li><li>有机硅垫片被切断使用剪影SD刀具的。这使我们能够精细地控制用于控制细胞的生长期间的流动速率和剪切应力的流动腔室的尺寸。通常情况下，一个信道被切断6.4毫米宽了19毫米，长为0.4mm的硅树脂的片材。的流量所需的属特1.0帕（10达因/厘米2）的剪切应力的计算假设层流中的矩形通道与方程： </li></ol><img alt="式（1）" fo:content-width="0.737in" fo:src="/files/ftp_upload/50163/50163eq1highres.jpg" src="/files/ftp_upload/50163/50163eq1.jpg" /> 其中 Q是流率，τ是剪切应力，μ是介质的粘度，这里假定为1.0毫帕（0.01达因*秒/ cm 2），h是高度和w是流动室的宽度。 <ol start="3"><li>的流动腔室的顶部件对齐的垫圈中的细胞培养皿中，并与磁性环固定。该组件填充用异丙醇（IPA）进行灭菌。 </li><li>全流系统的组装。细胞培养皿中的流通口连接到三通阀。连接打开的阀门30ml注射器第IPA冲洗通过该系统，然后用4％的胎儿小牛血清（FCS）的McCoy的培养基用30ml洗涤。该系统，然后用20毫升的V​​EC技术细胞生长培养基填充。盖被放置在顶部的注射器。捕捞水库注射器的帽针在移动中储饲料。不育0.2μm的过滤器被安装在盖的进气口，以防止系统的污染。流动腔细胞接种的准备。 </li></ol> 1.2细胞培养<ol><li>人脐静脉内皮细胞（HUVEC）和生长培养基购自VEC技术（伦斯勒，NY），并生长至汇合在T25药剂。 </li><li>除去生长培养基中，从烧瓶中，并用2毫升2.5％的胰蛋白酶释放的细胞单层。一旦细胞是在溶液中，向烧瓶中的细胞培养液中加入10毫升的胰蛋白酶消化，骤冷。 </li><li>钍E细胞悬浮液离心5分钟，并除去上清液。将细胞重新悬浮在1ml注入流动室的细胞培养基（含血清）。 </li><li>将细胞悬浮液（0.5毫升，约50,000个细胞）被加载到一个注射器中，并注入流动室通过一个三通阀。 </li><li>细胞被允许定居，并坚持到玻璃基板流开始前2小时。在细胞生长根据流的培养箱中在37℃，进行1〜5天，直到汇合。 </li></ol> 1.3悬臂的制备和细胞压痕<ol><li>无针尖AFM的悬臂（纳米世界，瑞士）在硝酸中清洗5分钟，并用氨基丙基三乙氧基硅烷（APTES）在蒸镀室官能。 </li><li> 5毫克/毫升（重量）NHS-磺基-LC-生物素的Hank缓冲盐溶液（HBSS）中制备的溶液。在悬臂被淹没在溶液中15分钟，以共轭门硅烷N-羟基琥珀酰亚胺（NHS）化学。 </li><li> VEC技术细胞培养介质（包括血清）的20毫升培养12小时streptavadin珠用200μl的溶液是由无生物素培养基中。在珠，用磁铁从培养基中除去，并通过0.22μm的无菌过滤器过滤介质。 </li><li>流动室除去从细胞培养皿中，并在37℃的无生物素培养基将细胞洗涤。 </li><li> 1微升2.4μm的珠粒涂有链霉抗生物素蛋白在1ml无生物素培养基中的储备溶液制备，并加入100微升的股票的细胞培养皿中。 </li><li>链霉亲和素珠被拾起的悬臂通过登陆在玻璃表面上的胎圈，缩回旁边的前端，在胎圈定位在悬臂的先端，然后向下按压悬臂到胎圈和休息的几秒钟。 </li><li>在悬臂的时间测量的灵敏度d的缩进到裸露的玻璃的区域和使用的曲线的斜率设置作为电压的函数的尖端偏转。 </li><li>在悬臂的弹簧常数，然后计算热校准在MFP3D软件。 </li><li>然后将校准悬臂用于缩进的样品，如在图2中示出。这些2.4微米珠提供了一个较大的接触面积，使与细胞表面的软糖萼层的机械性能，可以检测。该悬臂被定位在附近的细胞核的细胞到细胞里的前端用来设置上方约3微米，在细胞表面的悬臂高度和柔软的方法。该软件被设置为20个重复的凹槽7 nN的一个最大的力为1μm/秒的速率。约6秒之间经过连续接触。这是可能使用不同速率的压痕测试的糖复合物的时间依赖特性，虽然在这些初始<html>实验中，只有一个单一的压痕率（1微米/秒）被使用。 </li></ol> 2。压痕理论到的弹性半空间与半径为R的球中的压痕可以描述采用Hertz理论缩进的力，F由下式给出： <img alt="公式2" fo:content-width="1.157in" fo:src="/files/ftp_upload/50163/50163eq2highres.jpg" src="/files/ftp_upload/50163/50163eq2.jpg" /> 其中δ是压痕深度和 E *是根据测试（ 图3）的材料的弹性模量降低。以一个无限刚性的压头撞击均匀的弹性半空间的情况下，E *是由下式给出： 3/50163eq3.jpg“/&gt; 其中 E是弹性模量，ν是材料的泊松比。与聚合物薄膜的最近的工作已激发用于确定的模量和厚度的薄膜1的两个层模型的发展。我们应用此模型细胞生物学处理作为电池主体的表面上的均匀的薄的软膜糖萼。使用此模型中，该系统的模量的减少变为： <img alt="式（4）" fo:content-width="2.85in" fo:src="/files/ftp_upload/50163/50163eq4highres.jpg" src="/files/ftp_upload/50163/50163eq4.jpg" /> 其中 ，E GC是的蛋白质复合物的弹性模量，E 细胞的细胞体的模量，P，Q和n是已经凭经验确定从聚合物中配合， 而 z是由下式给出的常数： 其中 t是的蛋白质复合物层的厚度。这些参数的示意图如图3所示。该模型已被证明是更严厉的基板1上的薄膜的弹性模量和厚度的确定一个准确的方法。这个方程可以使用，以适应从缩进到细胞得到的曲线，以确定的模量和厚度的内皮糖萼，如在图4中示出。

<ol>
	<li>Clifford, C., Seah, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoindentation+measurement+of+young's+modulus+for+compliant+layers+on+stiffer+substrates+including+the+effect+of+poisson's+ratios.">Nanoindentation measurement of young's modulus for compliant layers on stiffer substrates including the effect of poisson's ratios.</a> Nanotechnology. , (2009).</li><li>Gouverneur, M., Spaan, J. A. E., Pannekoek, H., Fontijn, R. D., Vink, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+stimulates+incorporation+of+hyaluronan+into+endothelial+cell+glycocalyx.">Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx.</a> Am. J. Physiol. Heart. Circ. Physiol. 290 (1), 458-452 (2006).</li><li>Hocde, S. A., Hyrien, O., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+distribution+relative+to+neutrophil+surface+topography+assessed+by+tirfm.">Cell adhesion molecule distribution relative to neutrophil surface topography assessed by tirfm.</a> Biophysical Journal. 97 (1), 379-387 (2009).</li><li>Lipowski, H. H. <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+glycocalyx+as+a+barrier+to+leukocyte+adhesion+and+its+mediation+by+extracellular+proteases.">The endothelial glycocalyx as a barrier to leukocyte adhesion and its mediation by extracellular proteases.</a> Annals of biomedical engineering. 40 (4), 840-848 (2012).</li><li>Lu, L., Oswald, S. J., Ngu, H., Yin, F. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+actin+stress+fibers+in+living+cells.">Mechanical properties of actin stress fibers in living cells.</a> Biophysical Journal. 95 (12), 6060-6071 (2008).</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 Archiv. European Journal of Physiology. 440 (5), 653-666 (2000).</li><li>Spillmann, C. M., Lomakina, E., Waugh, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+adhesive+contact+dependence+on+impingement+force.">Neutrophil adhesive contact dependence on impingement force.</a> Biophysical Journal. 87 (6), 4237-4245 (2004).</li><li>vanden Berg, B. M., Vink, H., Spaan, J. A. E. <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+glycocalyx+protects+against+myocardial+edema.">The endothelial glycocalyx protects against myocardial edema.</a> Circulation Research. 92 (6), 592-594 (2003).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+impact+of+membrane+microtopology+on+effective+two-dimensional+affinity.">Quantifying the impact of membrane microtopology on effective two-dimensional affinity.</a> J. Biol. Chem. 276 (16), 13283-138 (2001).</li><li>Vink, H., Duling, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Distinct+Luminal+Domains+for+Macromolecules,+Erythrocytes,+and+Leukocytes+Within+Mammalian+Capillaries.">Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries.</a> Circulation Research. 79, 581-589 (1996).</li></ol>

没有利益冲突的声明。

我们使用从两层模型和Hertz理论建模的循环血液中的白细胞与血管内皮壁的相互作用的计算出的值。我们计算一个微绒毛上与10 PN负载下的直径为50nm的白细胞会缩进约150nm到糖萼，只有一小部分的总厚度。这表明，糖复合物，与如在本实验中测得的性能，是一种显着的细胞 - 细胞相互作用的障碍，并可以是一个大的空间位阻的细胞期间必须克服的白细胞粘附的粘附级联期间。 ?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>麦考伊的中</td><td> Gibco公司</td><td> 16600-082 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30070 </td><td></td></tr><tr><td>血管内皮细胞生长培养基</td><td>血管内皮细胞技术</td><td> MCDB-131 </td><td></td></tr><tr><td>汇集人类脐静脉内皮细胞</td><td>血管内皮细胞技术</td><td> PHUVEC/T-25 </td><td></td></tr><tr><td>硫酸</td><td> JT贝克</td><td> 9681-02 </td><td></td></tr><tr><td>过氧化氢</td><td> VWR </td><td> BDH3742-1 </td><td></td></tr><tr><td> （3 - 氨基丙基）三乙氧基硅烷</td><td> Aldrich公司</td><td> 440140-100ML </td><td></td></tr><tr><td>异丙醇</td><td> VWR </td><td> BDH8999-4 </td><td></td></tr><tr><td>胰蛋白酶</td><td> Cellgro </td><td> 25-054-C1 </td><td></td></tr><tr><td>汉克的缓冲盐溶液</td><td> Gibco公司</td><td> 14175-095 </td><td></td></tr><tr><td>磺基-NHS-LC-生物素</td><td> Thermo Scientific的</td><td> 21335 </td><td></td></tr><tr><td> Streptavadin珠</td><td>磁珠</td><td> 112.06D </td><td></td></tr><tr><td> MFP-3D原子力显微镜</td><td>庇护研究</td><td></td><td></td></tr><tr><td>无针尖悬臂</td><td>纳米世界</td><td> ARROW-TL1-50 </td><td></td></tr><tr><td>剪影SD </td><td> Quickutz </td><td>剪影-SD </td><td></td></tr><tr><td>硅橡胶</td><td>斯托克韦尔弹性体</td><td> SE50-RS </td><td></td></tr><tr><td> 30毫升注射器</TD&gt; <td>本顿迪金森</td><td> 309650 </td><td></td></tr><tr><td> 18号针</td><td>本顿迪金森</td><td> 305196 </td><td></td></tr><tr><td>拓</td><td> Hospira公司</td><td> 4429-48 </td><td></td></tr><tr><td> 4单向阀</td><td>泰利福</td><td> W21372 </td><td></td></tr><tr><td>男/女港口上限</td><td>史密斯医疗</td><td> MX491B </td><td></td></tr><tr><td>蠕动泵</td><td> Watson-Marlow公司</td><td> 401U / D </td><td></td></tr><tr><td>蠕动管</td><td> Watson-Marlow公司</td><td> 903.0016.016 </td><td></td></tr><tr><td>无菌过滤器</td><td>颇尔生命科学</td><td> 4652 </td><td></td></tr></tbody></table>

quantifying the mechanical properties of the endothelial glycocalyx with atomic force microscopy

我们理解白细胞捕获期间的白细胞与血管壁的相互作用的限制由内皮表面层的机械性能的一个不完整的理解。它是已知的非均匀分布的白细胞粘附分子相对于表面拓朴图3，该地形限制粘结形成其他的表面9，并且，生理上的接触力（≈5.0 - ：10.0，PN每微绒毛）可以为压缩的微绒毛少三分之一其休息的长度，增加分子的可访问性的相对表面3，7。我们认为，血管内皮细胞的两层结构，相对刚性的单元主体，加上糖萼腔表面6上，一个软保护糖衣。它已被证明，糖复合物可以作为阻挡减少白细胞粘附于内皮表面4。在这份报告中，我们着手解决的内皮细胞表面的变形，以了解血管内皮的机械刚度，可能会影响债券的形成。静态培养中生长的血管内皮细胞不表达一个强大的糖复合物，但在生理流动条件下生长的细胞开始观察体内的糖复合物。内皮细胞体的模量，已使用原子力显微镜（AFM）测定的，是大约5到20千帕5。的糖复合物中的厚度和结构进行了研究，使用电子显微镜8，和已糖复合物的模量的近似使用间接的方法，但就我们所知，没有已经发表在活细胞中的蛋白质复合物的弹性模量的直接测量的报告。在这项研究中，我们提出了一个新的原子力显微镜探针对细胞已培养的条件下，最大限度地发挥他们的糖被表达马压痕实验科的直接测量的弹性模量和厚度的内皮糖萼。

在一个典型的实验中，从一个给定区域的细胞得到的20的力与距离曲线，通常在核周区域，靠近，但不包括在核（〜2μm的范围内）。曲线，考虑到对准测量的持续时间的任何样品漂移，然后取平均值，以除去悬臂噪音，如在图4中示出。曲线进行了分析和合适的被开发用于确定的弹性模量和厚度的薄的聚合物膜1的两个层模型。从拟合的曲线的25个细胞中，我们已经确定，管腔?...

Watch this Scientific Journal Video about Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy at JoVE.com

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

内皮糖萼的机械特性测定压痕使用AFM的悬臂上的微米大小的球体。内皮细胞培养在一个自定义的腔室，在生理流动条件下，诱导糖萼表达。使用薄膜的模型，以确定具有糖萼厚度和弹性模量的数据进行了分析。

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 ...

quantifying-mechanical-properties-endothelial-glycocalyx-with-atomic

Research

JoVE Journal

Bioengineering

13.1K 次观看.  University of Rochester. 内皮糖萼的机械特性测定压痕使用AFM的悬臂上的微米大小的球体。内皮细胞培养在一个自定义的腔室，在生理流动条件下，诱导糖萼表达。使用薄膜的模型，以确定具有糖萼厚度和弹性模量的数据进行了分析。

视频: Quantifying the Mechanical Properties of the Endothelial Glycocalyx with 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 ...

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, ...

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 ...

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 ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

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 ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...