This research is supported by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Programme, the Start-up Grants (M4330002.C70) from Nanyang Technological University, and AcRF Tier 2 (MOE2014-T2-2-172) from Ministry of Education, Singapore. The authors thank Joey Yam Kuok Hoong for participating in the demonstration of this protocol.

1.生物膜栽培<ol><li>细菌菌株制备<ol><li>前1天生物膜培养，接种2毫升合适的生长培养基从冷冻的细菌培养制备的浮游细菌培养物。使用的Luria肉汤培养基（10克L -1 NaCl的10克L- -1酵母提取物，和10g L -1蛋白胨）为粘液P.铜绿假单胞菌和Δ 像素和ΔPSL缺陷的突变体。孵育过夜，在37℃和200 rpm振摇条件。稀释过夜培养物，以使用分光光度计的OD 600的0.40。 </li><li>装配流动池设置，已经描述先前14，最好是在移动台或台车，可以被带入显微镜室成像流动池无需拆卸。 </li><li>在2升或5升瓶的每个流动池准备无菌生长培养基中。使用最少的培养基M9（48毫的 Na 2 HPO 4，22毫KH 2 PO 4，19 mM的氯化铵 ，9毫摩尔NaCl，2mM的硫酸镁 ，和0.1mM的CaCl 2）补充有0.04％葡萄糖（重量/体积）和0.2％（重量/体积）酪蛋白氨基酸）的P.铜绿假单胞菌流细胞生物膜。 </li><li>分装荧光微球进入离心管，并在无菌H 2 O的5分钟，在离心机降速为9,391.2克除去上清，重悬在1ml的生长培养基以除去叠氮化钠（消毒剂）添加到生长培养基在2L或5L的介质瓶之前。 注意：不同尺寸的微球有不同的浓度，应作相应稀释到指示。例如，分散120微升1.0微米直径的微球，15微升0.5微米直径的微球和0.96微升0.2微米直径的微球体的成2升生长培养基，得到2.18×10 6个微球毫升-1每个粒子大小。 </li><li>安装介质瓶upst流动池的铰孔和允许生长培养基流过整个设置。停止流动，并注入稀释过夜培养成流动池室。使细菌持续流动生长培养基以约5.5×10 -3米秒 -1，或8的转速用蠕动泵的流速之前附加到盖玻片基底1小时。 注：生物膜通常生长在室温（25℃）下3-7天。 </li><li>另外，对于静止培养设置，通过加入10微升过夜培养物到1ml生长培养基与颗粒稀释过夜培养100倍在微量离心管中。大约500倍相比，用于流量生物膜增加颗粒浓度在静态培养生长培养基（例如，清洗并重悬600微升1.0微米直径球在10ml生长培养基），为流动池生物膜不断地与补充粒子，但静态的文化不是。 <ol><li>加入2001，L稀释过夜培养具有颗粒8以及幻灯片室。在37℃静态条件下孵育。生物膜通常生长1天。每日与颗粒新鲜生长培养基更换废生长培养基如果生物膜生长超过1天。 </li></ol></li></ol></li></ol> 2.显微镜<ol><li>在第3天及5中，关闭流量从蠕动泵和使流通池设置成显微镜室。附近的流动池室的入口和出口夹紧管道，以防止漂移从流量（通过在环境中的变化的系统误差的原因）。将流动池到直立显微镜的显微镜载物台。 注：使用倒置显微镜的微孔成像。 </li><li>使用荧光显微镜用40X油浸物镜采取微球运动的嵌入在生物膜在各个位置（小菌落和平坦未分化层）（第3天及5），用于视频和在不同的日子时间和空间的调查。以短视频具有更高的帧速率，以调查在短的时间尺度上发生的事件（ 如 1.5-3分钟视频以每秒25-50帧的快速动态的帧速率），以及更长的视频以较低的帧速率来调查过较长时间的事件尺度（ 如 15-30分钟的视频在2.5-5帧/秒的速度较慢动态帧速率）。 注：视频通常包含5-10个粒子，一般是1.0〜2.5 GB的文件大小。较大的菌落可以容纳更多的颗粒。以5-10的影片为每个类别（平层分化不同的地点不同的菌落和如 5-10视频）。生物膜具有异质结构，可能包括小菌落，通道，空隙和平面未分化层。 </li><li>保存为相应格式的视频，可以通过斐济/ ImageJ的读取（ 如 CZI，TIF）。 </li><li>检查视频漂移通过成品VID滚动EO和观察到的颗粒不会在相同的方向同时移动。漂移可以通过z台运动，在流动池中的电流，抗振动台，空气压力或温度变化的不平衡引起的。使用通常设有显微镜软件的后处理技术正确轻微漂移。丢弃任何影片，其中漂移不能纠正。记录以下参数，其中粒子跟踪分析过程中是必需的：分辨率（像素/ UM），帧数，持续时间。 </li><li>从显微镜阶段卸下流通池，并重新启动蠕动泵，继续培养生物膜。 </li></ol> 3.粒子跟踪分析<ol><li>通过使用插件TrackMate开源软件（ImageJ的）获取粒子轨迹。下载斐济在<a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji。</a>打开与斐济和在插件菜单中选择视频，请选择跟踪和TrackMate。 </li><li>检查或调整日的设置Ë窗口提示初始校准设置，记录在步骤2.4匹配视频参数。 </li><li>使用ImageJ在TrackMate检测颗粒。 <ol><li>选择“数检测器”，输入粒径和阈值（ 如 1000）。通过视频滚动而点击“预览”按钮，检查紫圆跟随整个视频的颗粒。调整直径和阈值作为必要的：提高阈值，如果紫色圆圈出现在空的空间。降低阈值，如果检测到没有足够的颗粒。点击下一步按钮即可完成检测的颗粒。 </li><li>与选项呈现给添加或删除的“初始阈值”检测到的粒子，“选择视图”和“选择过滤器”窗口，而不调整继续，如果初始粒子探测是令人满意的，当单击下一步按钮。 </li><li>在OPTI选择“简单LAP跟踪器”对吧“选择跟踪方法”窗口中的粒子很少迁出焦点生物膜，很容易跟踪。使用建议或低链接和缺口闭合的最大距离值，然后单击下一步。 </li><li>检查粒子跟踪是通过视频滚动地看到，粒子遵循TrackMate绘制的轨道，并没有多余的或丢失的轨道满意。跳过各种过滤步骤。进入到最后的窗口“中选择一个动作”。选择从下拉菜单中选择“导出曲目xml文件”，然后单击“执行”。选择一个文件夹保存的曲目。 注：有在公共领域的粒子运动轨迹和使用microrheology的分析提供各种方案。 msdanalyzer和TrackArt是，在Matlab环境下运行的粒子跟踪分析程序的示例。 </li></ol></li><li> Matlab的准备通过选择菜单导入粒子轨迹中的XML文件matlab文件&gt;设置路径...并添加脚本文件夹中与斐济包。 </li><li>为了分析的粒子轨迹与msdanalyzer，下载链接，压缩文件或tar.gz文件at <a href="http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories">http://www.mathworks.com/matlabcentral/fileexchange/40692-mean-square-displacement-analysis-of-particles-trajectories</a> . <ol><li>提取@msdanalyzer文件夹，15拖放一个属于Matlab的路径的文件夹中（ 例如C： Documents和Settings &lt;用户名&gt; 我的文档 Matlab的）。启动Matlab和启动分析仪通过键入： MA = msdanalyzer（2，&#39;嗯&#39;，&#39;秒&#39;） 其中微米和秒是物理的空间和时间单元中的视频。 <ol><li>导入粒子轨迹通过键入： [曲目，MD] = importTrackMateTracks（&#39;filename.xml中&#39;，&#39;clipZ“;，“scaleT”） MA = ma.addAll（轨道）; 其中“clipZ”删除Z尺寸为2D视频，和&#39;scaleT“。 </li><li>画出轨迹图上使用： ma.plotTracks; ma.labelPlotTracks; </li><li>计算使用粒子的MSDS： MA = ma.computeMSD; ma.msd </li><li>绘制每个粒子的MSDS： 数字 ma.plotMSD </li><li>通过重复3.5.1.1至3.5.1.4）组合来自同一categery的（嵌入在野生型小菌落在第3天，例如颗粒）的其他视频粒子轨迹。 </li><li>绘制集合平均或平均值的所有曲线： ma.plotMeanMSD 从线性的Y轴和X轴改变为对数刻度。在很长的滞后时间，MSD的曲线变得嘈杂，由于统计不足，在较长的时间尺度。 MSD的曲线也急剧上升，由于动态误差。曲线的这些区域可以通过使用刷子工具被移除选择曲线的末端，选择&#39;刷牙&#39;&gt;&#39;删除Unbrushed“在工具菜单中。 </li></ol></li><li>下载Ezyfit在<a href="http://www.fast.u-psud.fr/ezyfit/">http://www.fast.u-psud.fr/ezyfit/</a>并添加Ezyfit属于Matlab的路径的文件夹（ 如 C： Documents和Settings &lt;用户名&gt; 我的文档 Matlab的）。通过键入Matlab的工作空间efmenu安装安装Ezyfit菜单。重新启动的MATLAB与图中窗口Ezyfit菜单。通过选择Ezyfit菜单“显示适合&#39;&gt;&#39;力量&#39;&gt;&#39;A * X ^ N&#39;装上MSD曲线的功法是n是估计的扩散指数α。 注：msdanalyzer需要的曲线在Matlab拟合工具箱MSD曲线的拟合。 Ezyfit是一种替代和免费的软件，可以进行曲线拟合。 </li><li>清除当前粒子运动轨迹和MSDS来计算新粒子的MSD在其他地点或时间： 明确</LI&gt; <li>颗粒的介质（对照组）各平均MSD曲线和菌落和各种品系的未分化的区域，复制并粘贴曲线到一个图进行比较计算后。样品刚度和交联的增加与较低的MSD值。平曲线具有较低的α和样品比用更高的α陡峭的曲线更富有弹性。 </li><li>选择曲线进入“工具”来看看数据统计，如中位数和范围。胎圈的MSD正比于蠕变柔，J其中珠被根据关系嵌入材料（吨）， <img alt="式（1）" src="/files/ftp_upload/53093/53093eq1.jpg" /> 其中 J =蠕变柔，D =粒径，K B =玻尔兹曼常数 ，T =温度和 t =时间。 </li></ol></li></ol>

<ol>
	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms.">Microbial biofilms.</a> Annu Rev Microbiol. 49, 711-745 (1995).</li><li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat Rev Microbiol. 8, 623-633 (2010).</li><li>Yang, 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=Distinct+roles+of+extracellular+polymeric+substances+in+Pseudomonas+aeruginosa+biofilm+development.">Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development.</a> Environ Microbiol. 13, 1705-1717 (2011).</li><li>Chew, S. C., 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=Dynamic+Remodeling+of+Microbial+Biofilms+by+Functionally+Distinct+Exopolysaccharides.">Dynamic Remodeling of Microbial Biofilms by Functionally Distinct Exopolysaccharides.</a> mBio. 5 (4), (2014).</li><li>Irie, Y., 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=Self-produced+exopolysaccharide+is+a+signal+that+stimulates+biofilm+formation+in+Pseudomonas+aeruginosa.">Self-produced exopolysaccharide is a signal that stimulates biofilm formation in Pseudomonas aeruginosa.</a> Proc Natl Acad Sci U S A. 109, 20632-20636 (2012).</li><li>Zhao, 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=Psl+trails+guide+exploration+and+microcolony+formation+in+Pseudomonas+aeruginosa+biofilms.">Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms.</a> Nature. 497, 388-391 (2013).</li><li>Yang, L., Nilsson, M., Gjermansen, M., Givskov, M., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyoverdine+and+PQS+mediated+subpopulation+interactions+involved+in+Pseudomonas+aeruginosa+biofilm+formation.">Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation.</a> Mol Microbiol. 74, 1380-1392 (2009).</li><li>Yang, 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=Pattern+differentiation+in+co-culture+biofilms+formed+by+Staphylococcus+aureus+and+Pseudomonas+aeruginosa.">Pattern differentiation in co-culture biofilms formed by Staphylococcus aureus and Pseudomonas aeruginosa.</a> FEMS Immunol Med Microbiol. 62, 339-347 (2011).</li><li>Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+involved+in+matrix+formation+in+Pseudomonas+aeruginosa+PA14+biofilms.">Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms.</a> Mol Microbiol. 51, 675-690 (2004).</li><li>Bokranz, W., Wang, X., Tsch&#228;pe, H., R&#246;mling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+cellulose+and+curli+fimbriae+by+Escherichia+coli+isolated+from+the+gastrointestinal+tract.">Expression of cellulose and curli fimbriae by Escherichia coli isolated from the gastrointestinal tract.</a> J Med Microbiol. 54, 1171-1182 (2005).</li><li>Denkhaus, E., Meisen, S., Telgheder, U., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+methods+for+characterisation+of+biofilms.">Chemical and physical methods for characterisation of biofilms.</a> Microchim Acta. 158, 1-27 (2007).</li><li>Waigh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrheology+of+complex+fluids.">Microrheology of complex fluids.</a> Rep Prog Phys. 68, 685-742 (2005).</li><li>Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-Tracking+Microrheology+of+Living+Cells:+Principles+and+Applications.">Particle-Tracking Microrheology of Living Cells: Principles and Applications.</a> Annu Rev Biophys. 38, 301-326 (2009).</li><li>Weiss Nielsen, M., Sternberg, C., Molin, S., Regenberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa.+and+Saccharomyces+cerevisiae.+Biofilm+in+Flow+Cells.">Pseudomonas aeruginosa. and Saccharomyces cerevisiae. Biofilm in Flow Cells.</a> J Vis Exp. , e2383 (2011).</li><li>Tarantino, N., 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=TNF+and+IL-1+exhibit+distinct+ubiquitin+requirements+for+inducing+NEMO-IKK+supramolecular+structures.">TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO-IKK supramolecular structures.</a> Journal of Cell Biology. 204, 231-245 (2014).</li><li>Yang, 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=Polysaccharides+serve+as+scaffold+of+biofilms+formed+by+mucoid+Pseudomonas+aeruginosa.">Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa.</a> FEMS Immunol Med Microbiol. 65, 366-376 (2012).</li><li>Gjermansen, M., Nilsson, M., Yang, L., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+starvation-induced+dispersion+in+Pseudomonas+putida+biofilms:+genetic+elements+and+molecular+mechanisms.">Characterization of starvation-induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms.</a> Mol Microbiol. 75, 815-826 (2010).</li><li>Qin, Z., 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=Role+of+autolysin-mediated+DNA+release+in+biofilm+formation+of+Staphylococcus+epidermidis.">Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis.</a> Microbiology. 153, 2083-2092 (2007).</li><li>Stoodley, P., 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+influence+of+fluid+shear+and+AlCl3+on+the+material+properties+of+Pseudomonas+aeruginosa.+PAO1+and+Desulfovibrio+sp.+EX265+biofilms.">The influence of fluid shear and AlCl3 on the material properties of Pseudomonas aeruginosa. PAO1 and Desulfovibrio sp. EX265 biofilms.</a> Water Science &amp; Technology. 43, 113-120 (2001).</li><li>J&#228;ger-Z&#252;rn, I., Grubert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podostemaceae+depend+on+sticky+biofilms+with+respect+to+attachment+to+rocks+in+waterfalls.">Podostemaceae depend on sticky biofilms with respect to attachment to rocks in waterfalls.</a> International Journal of Plant Sciences. 161, 599-607 (2000).</li><li>Matysik, A., Kraut, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TrackArt:+the+user+friendly+interface+for+single+molecule+tracking+data+analysis+and+simulation+applied+to+complex+diffusion+in+mica+supported+lipid+bilayers.">TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.</a> BMC Research Notes. 7 (1), 274-283 (2014).</li></ol>

The authors have nothing to disclose.

Microrheology is a useful tool for local rheological measurements in heterogeneous systems, such as microbial biofilms. It is a non-destructive technique, enabling the real-time monitoring of rheological changes within the same biological sample over multiple time points. In this protocol, particle-tracking microrheology was applied to Pel and Psl exopolysaccharide mutants in order to investigate how they affect the elasticity and effective crosslinking of the biofilm matrix. Psl favors the development of elastic biofilm...

大多数细菌细胞能够同时使用浮游（自由生活的），并生长1面接合（固着）模式。在成长的表面附着模式下，细菌细胞分泌，并包住自己在大量的胞外聚合物（EPS），形成生物膜。所述EPS主要由蛋白质，胞外多糖，胞外DNA，并且对生物膜形成2。它作为一个物理支架通过该细菌可以用于区分空间上和防止有害的环境条件和宿主反应的细菌。 EPS为不同的组件有生物膜形成3和改变EPS成分的表达不同的角色，可以极大地重塑生物膜结构4。车部件也可以作为信号分子5，最近的研究已经显示出一定的EPS部件与微生物细胞相互作用来引导它们的迁移和生物膜的differentiation 6-8。 研究EPS显着促进了基于由突变体缺陷在EPS 9,10的特定组件所产生生物膜的形态分析。此外，EPS的特点通常是在宏观尺度（散装特征）11。形态学分析可以然而缺乏定量细节和散装表征，它返回平均值，失去生物膜的异质性内存在的细节。现在有越来越多的趋势发展到了EPS的力学性能的实时特性，在微观尺度。这个协议演示如何粒子追踪microrheology能够确定的基质组分贝利上的铜绿假单胞菌生物膜4的粘弹性和有效交联的时空效果和PSL胞外多糖。 被动microrheology是简单且廉价的rh的eology方法，提供了通过的材料制成的空间微观流变采样的最高迄今为止12,13。在被动microrheology，探针球体放置在样品中与他们的布朗运动，通过热能量（K（B T））驱动的后跟视频显微镜。几个颗粒可以同时被跟踪，和颗粒的时间依赖性坐标遵循常规随机游走。因此，平均来说，粒子保持在同一位置。然而，位移的标准偏差或均方的颗粒位移（MSD），不为零。由于粘性流体流动，在粘性流体粒子MSD线性增长随着时间的推移。与此相反，聚合物的交联存在于粘弹性或弹性物质有助于他们抵抗流动，颗粒变得有限在他们的位移，导致在MSD曲线（图1A）的高原。这个观察如下的关系MSDαt <s向上&gt;α，其中 ，α是相关的物质的弹性和粘性的贡献率的扩散指数。为粒子朝着粘性流体α= 1，在粘弹性物质0 &lt;α&lt;1，并且在弹性物质α= 0。MSD还可以用于计算蠕变柔量，它是该材料的倾向永久过度变形时间并估计多么容易的材料价差。 颗粒的尺寸，密度和表面化学是微观流变试验的正确应用关键和被选择相对于所研究的系统（在此情况下，生物膜基质的聚合物中，参见图1B）。首先，粒子测量物质与结构，是比颗粒本身小得多的流变性。如果该物质的结构是相似的规模到颗粒，票面的运动视察由各个结构的形状和取向扰动。然而，如果包围粒子的结构是要小得多，这效果小，平均掉，呈现均匀的环境到粒子（图1B）。其次，该粒子的密度应类似于介质（1.05克毫升-1用于水性介质），使得沉降避免和惯性力是可以忽略不计。大多数颗粒聚苯乙烯晶格符合上述标准。理想的情况下，该颗粒不与生物膜基质的聚合物相互作用作为粒子的MSD的流变解释才有效，如果运动是随机的，具有物质结构从动通过热能和碰撞。这可以通过检查是否在探头颗粒趋于结合或反弹的预生长的生物膜的表面被观察到。然而，尽管缺乏吸引力的生物膜中，颗粒必须能够被并入基质。另外，生物膜的物理化学异质性可能会导致不同的颗粒的更适合作为在生物膜的不同区域的探针。因此，不同大小和表面化学的颗粒应被应用到生物膜。 这样，粒子MSD是能够提供关于组件如何不同向流变性和生物膜的传播有用信息。此外，使用不同的探针的允许一个派生对生物膜的空间异质性理化信息。此方法可用于测试作用抗微生物处理的生物膜的机械性能，或施加到混合物种的生物膜研究如何生物膜的机械性能从引入另一物种的改变。粒子的MSD还可以是用于表征生物膜分散有用。这样的研究将有助于我们生物膜的理解，有可能提高生物膜处理一个生物膜的有益活动的第二工程。

<table>
	<tbody>
		<tr>
			<td>Fluorspheres</td>
			<td>Invitrogen</td>
			<td>F-8821</td>
			<td>1.0 um red fluorescent (580/605) microspheres with carboxylate modification</td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager M1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Epifluorescent Microscope</td>
		</tr>
		<tr>
			<td>Masterflex L/S Digital Drive 07523-80</td>
			<td>Cole-Parmer</td>
			<td>EW-07523-80</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr>
			<td>Flow Cell Chambers</td>
			<td colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Bubble Trap</td>
			<td colspan="3">Technical University of Denmark</td>
		</tr>
		<tr>
			<td>Silicone Tubing</td>
			<td>Dow Corning</td>
			<td></td>
			<td>3 mm outer diameter, 1 mm inner diameter</td>
		</tr>
		<tr>
			<td>Clear polypropylene plastic connectors&#160;</td>
			<td>Cole Parmer</td>
			<td>06365-83</td>
			<td>1/16 in. (1.588 mm)</td>
		</tr>
		<tr>
			<td>Binder Clips</td>
			<td></td>
			<td></td>
			<td>To clamp tubing</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td colspan="2">Thermo Scientific&#8482; Nunc&#8482;</td>
			<td>50 x 24 mm</td>
		</tr>
		<tr>
			<td>Syringe 3 mL</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2L Storage/Media Bottles</td>
			<td>VWR&#174;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trolley</td>
			<td></td>
			<td></td>
			<td>To hold biofilm setup</td>
		</tr>
	</tbody>
</table>

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.

The local viscoelastic properties of the biofilm in different regions of the biofilm, which included the voids (medium above the biofilm), plains (undifferentiated flat layer of cells) and microcolonies (see labels in Figure 2A) were investigated. The temporal changes in viscoelastic properties of the biofilm during maturation from days 3 to 5 were also determined. The MSD of the particles in the voids was used as a control and comparable to the MSD of particles in pure medium. In contrast, particles tra...

Watch this Scientific Journal Video about In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology at JoVE.com

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 ...

in-situ-mapping-mechanical-properties-biofilms-particle-tracking

Research

JoVE Journal

Bioengineering

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

9.6K 次观看.  Nanyang Technological University. 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.

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

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

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 ...

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, ...

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in these structures are held together by physical interactions mediated by a network of extracellular polymeric substances. Bacterial biofilms impact many human activities and the understanding of their properties is crucial for a better control of their development &#8212; maintenance or eradication &#8212; depending on their adverse or beneficial outcome. This paper describes a novel methodology aiming to measure in situ the local physical properties of the biofilm that had been, until now, examined only from a macroscopic and homogeneous material perspective. The experiment described here&#160;involves introducing magnetic particles into a growing biofilm to seed local probes that can be remotely actuated without disturbing the structural properties of the biofilm. Dedicated magnetic tweezers were developed to exert a defined force on each particle embedded in the biofilm. The setup is mounted on the stage of a microscope to enable the&#160;recording of time-lapse images of the particle-pulling period. The particle trajectories are then extracted from the pulling sequence and the local viscoelastic parameters are derived from each particle displacement curve, thereby providing the 3D-spatial distribution of the parameters. Gaining insights into the biofilm mechanical profile is essential from an engineer&#39;s point of view for biofilm control purposes but also from a fundamental perspective to clarify the relationship between the architectural properties and the specific biology of these structures.

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.

测微探针进行远程磁驱动<em&gt;原位</em&gt; 3D细菌生物膜物理性质的映射

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit 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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

一种实时3D 单粒子跟踪协议

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...