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>

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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 biofilms with high effective crosslinking, whereas Pel favors viscoelastic and looser biofilms. When both exopolysaccharides are produced, the biofilm microcolonies becomes less effectively crosslinked as the biofilm matures, consistent with a decrease in Psl over Pel.
Biofilms formed by different bacterial species have distinct EPS compositions. The EPS of mucoid P. aeruginosa strains used in this study mainly consists of exopolysaccharides 16. Other species may use large extracellular adhesion proteins 17 and DNA 18 as their major components of the EPS. The approach described here could be used to determine the rheological contribution of other EPS components and their effect on growth. In addition, biofilms grown in different setups may have drastically different physical properties that affect their rheology. For example, mucoid P. aeruginosa supplemented with glucose forms more biofilm with highly differentiated structures under flow compared to static conditions. Studies have also shown that biofilms exposed to shear stress such as flow makes the biofilms more cohesive and elastic 19,20. Important steps and factors to consider for successful biofilm particle-tracking microrheology study are as follows:
The system dimensions for investigation or scale of interest with respect to time and space
When considering spatial scales, one must consider the size of the structures that give rise to the viscoelastic properties of the system (in our case, the polymers of the biofilm matrix). The polymers surrounding the probe particle should be much smaller in structure than the particle and present an isotropic environment to the particle. High-resolution imaging is required to capture small particle motion or vibrations, but will reduce the amount of area imaged for equivalent file size. When considering temporal scales, elastic biofilms that exhibit slow dynamics require taking videos over longer time scales (e.g. hr) and using low frame rates (e.g. min). Shorter videos can be taken for viscoelastic biofilms with fast dynamics (min), but require higher frame rates (sec or millisec). Using suitable resolution, frame rate, video duration for each experiment will keep file sizes low for tractable analysis.
Biofilm viscoelasticity, heterogeneity and dimensions
Particle motion may be undetectable in very elastic biofilms (MSD &#60;10-4). In such cases, active microrheological techniques that apply force to displace the particle are preferable. Rheological sampling of different regions of biofilm should be made to ensure the mechanical heterogeneity of the biofilm is captured. Heterogeneous morphology, however, is not necessarily a good indicator of mechanical heterogeneity, as despite homogeneous appearances the biofilm can be complex in rheology: &#916;psl biofilms are more homogeneous in morphology and delayed in differentiation, but have a complex and heterogeneous rheological profile. In contrast, &#916;pel biofilms have a heterogeneous appearance with well-differentiated microcolonies but a constant rheology throughout the biofilm. For biofilms with large microcolony dimensions (e.g. &#62;50 &#181;m in diameter and height), it may be meaningful study the rheological differences according to height (top and bottom of microcolonies), or distance from microcolony edge.
Selection of probe particles
As described above, the size of particles should be larger than the structures of the materials that give rise to its viscoelastic properties. In addition, particles with different surface chemistry may result in binding to different areas and EPS components within the biofilm. Thus the effects of particles with different surface chemistry should be explored so that biofilm heterogeneity can be considered. All probe particles should have a high zeta potential to minimize agglomeration. Particles that bind together cannot be used as accurate probes and should be excluded from analysis.
Minimization of drift
Drift can be caused by temperature or air pressure changes, microscope stage movement (usually in the z-direction), anti-vibration table imbalance and internal flow within the system. Drifts usually result in superdiffusion of the particle and &#945; &#62; 1. An &#945; &#62; 1 indicates that other than thermal energy, active processes are involved moving particle and passive microrheology is not applicable.
Proper biofilm culture maintenance
Flow cells must be properly assembled to prevent leakages and biofilms must be kept free from contamination. Biofilms can also be grown in other setups alternative to the flow cell, such as slides and microwells for static culturing conditions. An inverted microscope should be used for imaging of microwells.
In summary, particle tracking microrheology is a useful technique that can be employed using microscopes standard to a biological laboratory. In addition, programs involved in the calculation and analysis of particle MSDs is readily available in the public domain. For example, msdanalyzer and TrackArt 21 (<a href="http://www2.sbs.ntu.edu.sg/staff/rskraut/index.php/trackart">http://www2.sbs.ntu.edu.sg/staff/rskraut/index.php/trackart</a>) are programs that run in the Matlab environment. However, because the technique relies only on thermal energy to move the particles, it is unable to resolve between samples of higher elasticity. Active techniques, such as magnetic tweezing, apply a force on the particle to act on and move within the sample and would be able to determine the viscoelasticity of highly elastic samples.

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 &amp;#956;m 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>Technical University of Denmark</td><td></td><td></td></tr><tr><td>Bubble Trap</td><td>Technical University of Denmark</td><td></td><td></td></tr><tr><td>Silicone Tubing</td><td>Dow Corning</td><td></td><td>3 mm outer diameter, 1&amp;nbsp;mm inner diameter</td></tr><tr><td>Clear polypropylene plastic connectors&amp;nbsp;</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>Thermo Scientific&amp;trade; Nunc&amp;trade;</td><td>50 x 24 mm</td><td></td></tr><tr><td>Syringe 3 ml</td><td>Terumo</td><td></td><td></td></tr><tr><td>27 &amp;nbsp;G Needle</td><td>Terumo</td><td></td><td></td></tr><tr><td>2 &amp;nbsp;L Storage/Media Bottles</td><td>VWR&amp;reg;</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 trapped in the biofilm vibrated at fixed positions and MSD values ranged from those typical of viscoelastic materials to strongly elastic gels (Figure 3).
Mucoid P. aeruginosa wild-type and its &#916;pel mutant strains (Figure 2A and 2B) developed microcolonies scattered on a thin plain by day 3 in their biofilms. The plain on day 3 was too thin for the investigation of rheological properties. In both biofilms formed by the mucoid P. aeruginosa wild-type and &#916;pel strains, the MSD of particles in the day 3 microcolonies was independent of time for time lags of 0.1 sec to 10 sec (&#945; = 0), indicating that the microcolonies were elastic (Figure 4, Table 1). The median creep compliances calculated from the corresponding particle MSDs were 4.3 x 10-2 Pa-1 and 3.6 x 10-2 Pa-1 respectively. By day 5 the creep compliance of the microcolonies in mucoid P. aeruginosa wild-type strain increased to 2.5 x 10-1 Pa-1, indicating a reduction in effective crosslinking within the matrix. The day 5 microcolonies were still elastic with &#945; = 0. &#916;pel did not change in rheology from days 3 to 5. The plains in mucoid P. aeruginosa wild-type and &#916;pel strains were similar in elasticity (&#945; = 0) and effective crosslinking to day 3 microcolonies, which could be reflective of their maturity (undifferentiated structure).
Biofilms formed by &#916;psl mutant strain (Figure 2C) were less differentiated and delayed in development. This resulted in biofilms with thick plains that developed microcolonies after day 3. The MSD values showed that the biofilms were much less effectively crosslinked than P. aeruginosa wild-type and &#916;pel strains (Figure 4, Table 1). The plains were viscoelastic, with &#945; = 0.12 and 0.26 at days 3 and 5 respectively. The median creep compliance of day 3 and 5 plains was 1.0 Pa-1. When microcolonies had developed in day 5, the creep compliance had decreased to 2.8 x 10-1 Pa-1, indicating that a threshold crosslinking is required for microcolony differentiation. However, the creep compliance was still greater than the creep compliance of younger day 3 mucoid P. aeruginosa wild-type and &#916;pel microcolonies. The day 5 microcolonies were viscoelastic with &#945; = 0.34. Thus, the P. aeruginosa biofilm is elastic and highly crosslinked in the absence of Pel. In the absence of Psl, the biofilm became viscoelastic and less crosslinked. In the mature biofilm structures such as the microcolonies, expression of both Pel and Psl exopolysaccharides resulted in distinct rheological differences over time. The reduction in crosslinking could be due to the reduction of Psl to Pel exopolysaccharides in the biofilm matrix during maturation.
<img alt="Figure 1" src="/files/ftp_upload/53093/53093fig1.jpg" /> 
Figure 1: Particle tracking microrheology in a biofilm. (A) Gray circles depict particle MSD in a viscous substance, which grows linearly with time and &#945; = 1. Black triangles depict particle MSD of an elastic substance, which is independent of time and a = 0. (B) Diagram of probe particle vibrating in the EPS of the biofilm. The particle is modeled like a bacteria cell and is similar in diameter. The matrix polymers that envelop the particle are much smaller than the particle.
<img alt="Figure 2" src="/files/ftp_upload/53093/53093fig2.jpg" /> 
Figure 2: Perspective and sectional view of day 5 biofilms (green) by confocal microscopy after continuous feeding with 1.0 &#181;m (purple), 0.5 &#181;m (red) and 0.2 &#181;m (orange) particles with negatively charged carboxylated surface. Biofilms formed from (A) mucoid P. aeruginosa, and mutants (B) &#916;pel and (C) &#916;psl strains. Particles are incorporated in all regions of the biofilm. Examples of microcolonies, plains and voids are labeled in (A). Modified from Chew et al., mBio, 2014 4. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53093/53093fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53093/53093fig3.jpg" /> 
Figure 3: Particle trajectories. Comparison of a track of a particle executing Brownian motion in growth medium (multicolored) compared to tracks of particles vibrating in biofilm microcolonies (dark blue). The segmented colors indicate breaks in tracking due to particle moving out of focus in the z-plane.
<img alt="Figure 4" src="/files/ftp_upload/53093/53093fig4.jpg" /> 
Figure 4: MSDs of 1.0 &#181;m particles in biofilms. Mucoid P. aeruginosa is elastic and microcolonies are reduced in effective crosslinking from days 3 to 5; &#916;pel is elastic and does not change in rheology from days 3 to 5; &#916;psl is viscoelastic and mainly consists of plains that do not change significantly in rheology from days 3 to 5.
<table>
	<tbody>
		<tr>
			<td>Strain</td>
			<td>Day</td>
			<td>Region</td>
			<td>Median Creep Compliance (Pa-1)</td>
		</tr>
		<tr>
			<td>wild-type</td>
			<td>3 day</td>
			<td>Microcolony</td>
			<td>4.3 x 10-2</td>
		</tr>
		<tr>
			<td>wild-type</td>
			<td>5 day</td>
			<td>Microcolony</td>
			<td>2.5 x 10-1</td>
		</tr>
		<tr>
			<td>wild-type</td>
			<td>5 day</td>
			<td>Plain</td>
			<td>4.1 x 10-2</td>
		</tr>
		<tr>
			<td>&#916;pel</td>
			<td>3 day</td>
			<td>Microcolony</td>
			<td>3.6 x 10-2</td>
		</tr>
		<tr>
			<td>&#916;pel</td>
			<td>5 day</td>
			<td>Microcolony</td>
			<td>3.6 x 10-2</td>
		</tr>
		<tr>
			<td>&#916;pel</td>
			<td>5 day</td>
			<td>Plain</td>
			<td>&#160;3.2 x 10-2</td>
		</tr>
		<tr>
			<td>&#916;psl</td>
			<td>3 day</td>
			<td>Plain</td>
			<td>1.0 x 100</td>
		</tr>
		<tr>
			<td>&#916;psl</td>
			<td>5 day</td>
			<td>Plain</td>
			<td>1.0 x 100</td>
		</tr>
		<tr>
			<td>&#916;psl</td>
			<td>5 day</td>
			<td>Microcolony</td>
			<td>2.8 x 10-1</td>
		</tr>
	</tbody>
</table>
Table 1: Median creep compliances and estimated diffusive exponents of wild-type and mutant strains according to biofilm age and region.

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

Universidad San Sebastian

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

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

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

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

基质贴壁细胞的微管吸吮的估计单元刚度

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

科研

生物工程

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 ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

自动 3D 光学相干断层扫描，以在大型空间尺度上阐明生物膜变形

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

环境科学

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 ...

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

通过 Connexin 43 结构中断干扰内皮生物力学

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

生物化学

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

微流体和 Microrheology 相结合测定软物质在重复相变过程中的流变特性

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

工程学

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

研究多孔介质中生物堵塞的微流体平台

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

生物膜弹性特性的快速无损表征

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.

作者聚焦：使用光学相干弹性成像表征环境生物膜力学及其在废水处理中的应用

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

使用光学相干弹性成像量化环境生物膜的弹性特性

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances ...

在生物膜的力学性能原位制图粒子追踪Microrheology

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此视频中的章节

基质贴壁细胞的微管吸吮的估计单元刚度

测微探针进行远程磁驱动

自动 3D 光学相干断层扫描，以在大型空间尺度上阐明生物膜变形

细胞外基质刚性三维肿瘤模型的纵向测量用粒子跟踪Microrheology

通过 Connexin 43 结构中断干扰内皮生物力学

微流体和 Microrheology 相结合测定软物质在重复相变过程中的流变特性

方法表征的共同发展生物膜和生境异质性

研究多孔介质中生物堵塞的微流体平台

使用光学相干弹性成像量化环境生物膜的弹性特性

使用光学相干弹性成像量化环境生物膜的弹性特性