The authors sincerely thank the Natural Sciences and Engineering Research Council of Canada, the Ontario Ministry of Research and Innovation, and the Canada Foundation for Innovation for funding this study.

1.准备玻璃器皿和文化<ol><li>在继续进行这个实验，准备5％绵羊血琼脂（SBA）板种植MRSA。孵育SBA板放置24小时，在37℃。此时间之后，单一的，良好的分离的菌落应存在以用于后续接种到液体介质中。商店划线SBA板的单菌落，在4℃下进行1 - 2个月。 注:羊的血琼脂平板进来20之间含有预先制备的包装 - 根据制造商25板，并通常由用加入了5％w / v的羊的血液中的胰蛋白酶大豆琼脂基。 </li><li>制成500毫升的胰蛋白酶大豆肉汤（TSB）中补充有1％葡萄糖和0.1％的NaCl。在此之后，收集并清洗2玻璃试管。如果高温高压消毒的盖子是试管不可用，用铝箔作为上限。蒸压TSB和试管随着一箱1毫升枪头。 </li><li>在生物安全柜或接近面包连接刻录机，吸管5毫升以前蒸压TSB为蒸压试管。要小心，以确保有足够的时间已经给出，让TSB和试管冷却至室温。从以前条纹5％SBA板，接种单菌落到6毫升TSB肉汤。一种将包含MRSA，并且第二试管将被用作阴性对照，以确保无菌。 </li><li>取接种TSB试管，并放置在往复培养箱24小时，在37℃和在200rpm。在此时间后微生物的生长应该是在MRSA的试管表观而没有生长应该发生在控制试管。 </li><li>就拿1毫升24小时的文化和吸管为6毫升新鲜TSB。孵育在37℃下6小时以200rpm。 </li><li>吸取1毫升6小时亚文化到1.5 ml离心管。离心3分钟，在850×g下。除去上清液并在1ml去离子H 2 O的重新悬浮颗粒离心机，重复，并重新暂停。 </li></ol><ol><li>取不锈钢和金的AFM样品盘（20毫米和分别10毫米直径），并清洗每一侧用5ml去离子H 2 O的持样品盘在用钳子优势手，并使用亚优势手来吸取在样品圆盘的每一侧5毫升后在含20ml去离子水中，2 O然后超声1分钟的烧杯中洗两边，地点样品盘。 </li><li>超声用钳后，从烧杯中取出样品盘。放置的磁盘，使它们在对一个开放的培养皿的边缘上的60°角倾斜到纸巾上。使用培养皿的盖以覆盖干燥的磁盘。让磁盘，然后再继续充分干燥。干燥时间可以变化之间4 - 8小时。 </li><li>干燥后，用钳子把样品盘成一个培养皿。 <ol><li>任选官能化的样品盘的表面与任何材料（例如，胶原蛋白，透明质酸，银纳米粒子等 ）。 </li><li>对于这个实验，使用聚-L-赖氨酸涂覆的衬底表面。对于聚L-赖氨酸官能化，吸管200 - 400微升0.1％的聚L-赖氨酸（以H 2 O）到样品盘，并让孵育1小时，在室温下，在培养皿。 </li><li> 1小时后，用钳子保持样品盘，和洗涤用1毫升去离子H 2 O的让干燥的纸巾按步骤2.2描述。 </li></ol></li><li>拿2个洗再悬浮细胞和板200 - 400微升样品上磁盘的生物安全柜内。如果样本盘被涂覆有聚-L-赖氨酸，孵育0.5小时，在室温下进行。 </li><li>细胞在样品盘孵育后，轻轻洗掉样品盘用1毫升去离子H 2 O的让磁盘映像之前干通宵。 </li></ol> 3. KPFM成像注:在这个实验中，使用安捷伦5500系列ILM-AFM。 <ol><li>要启动AFM，打开计算机单元连同AFM的HEB和MAC III控制器。打开AFM成像软件（ 如安捷伦PicoView）。一定要选择最初ACAFM或任何正确的名称是在AFM成像间歇性接触模式。 </li><li>在优势手使用AFM镊子，并保持该弹簧加载的夹子保持件与次要手打开，放置一个KPFM悬臂进给头件。处理和传送的AFM头件，以原子力显微镜，因为这单元（至少在5500 ILM-AFM）时包含易碎压电晶体单元，其控制X，Y和Z扫描方向性要小心。 注意:在这种情况下，所用的KPFM悬臂分别Mikromasch导电DPE（低噪声）悬臂具有2.7牛顿/米8​​0千赫和弹簧常数的平均谐振频率。悬臂与这些谐振频率和弹簧常数被选择，因为它们易于使用。悬臂具有较大弹簧常数和较高的谐振频率，也可以用于成像。 </li><li>谨慎地装载头片插入AFM并连接相应的电线（在此情况下两根线需要被插入:激光和阶段提升电机）。 </li><li>激光对准到悬臂的尖端。有的单位包含一个摄像头功能，这使得更容易对齐。如果相机的功能是不存在于原子力显微镜，对准到悬臂刀片如下所述: <ol><li>旋转前端到后端的旋钮顺时针移动激光上层建筑悬臂芯片。当激光到达芯片它会被阻塞，将不再是可见的。仅转动旋钮几次。 </li><li>转动前到后的旋钮，直到激光光斑重新出现。现在的激光是在芯片的边缘。 </li><li>旋转左到右的旋钮来定位在悬臂的激光。由于激光经过悬臂就会消失，重新出现我ñ接二连三。激光点应该在磨砂玻璃罩，其中所述光电二极管单元将在后面坐可见。 </li><li>转前端到后端旋钮逆时针向下移动悬臂当场朝向尖端直到在毛玻璃的斑点消失。 </li><li>转前到后把手顺时针只稍稍顺序，以便它只是就座于悬臂刀片来定位激光。激光光斑将重新出现在毛玻璃。 注:这是激光定位方法跳水板悬臂。对于三角悬臂，校准过程稍有不同。 </li></ol></li><li>一旦激光对准，保持在"开启"位置的阶段提升电机10秒，以确保该悬臂和样品之间有足够的空间将所述样本到AFM时。 </li><li>将样品在KPFM样品阶段。接地用铜线将样品。从AFM到样品台连接的相应导线。连接S踏歌的AFM。在大多数情况下，该阶段是使用磁铁保持在适当位置。 </li><li>在AFM软件，调谐悬臂，然后开始在悬臂尖端到样品的方法。确保稳定时间被设定为不超过10毫秒，该方法的速度不超过2.0微米/秒，以避免尖的损伤。 </li><li>一旦悬臂刀片是在样品表面上，选择的成像窗口的大小和理想成像区。优化的I和P增益随着悬臂设定点，使得地形成像优化。 <ol><li>一旦地形成像进行了优化，打开KPFM模块（FM或AM模式;在这里，使用FM模式）。应当指出的是KPFM成像是5微米×5微米的成像窗口之外非常具有挑战性。此外，对于最佳KPFM成像，使用带有慢扫描速度一512×512分辨率（0.02 - 0.05行/秒）。 </li></ol></li><li>对于FM-KPFM设置（不ACAFM设置），设置5之间的驱动百分比 - 10％。设置悬臂FR1之间equency - 5 kHz的2 kHz的带宽。置的I和P增益为FM-KPFM设置到0.3％。 <ol><li>不同的信号带宽和I和样品表面之间的P涨幅。为了优化SP成像，进一步调整信号带宽和I和P的收益，以获得最佳的图像。 I和P收益的推荐范围是0.22 - 0.35％。 </li></ol></li><li>过程和使用后的图像处理软件来收集数据SP分析收集KPFM图像。 </li></ol>

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The authors have nothing to disclose.

KPFM被用作一种新的技术，用于获得表面电数据。它通常被用作研究化学电荷分布的方法和最近才开始被应用于对微观和纳米尺度的生物系统的研究。从收集到的数据，我们发现，微生物并未以容易地连接到清洗不锈钢和金表面，即使经过3小时的静态培养。聚-L-赖氨酸的官能表面呈30分钟后快速微生物附着。在功能化表面微生物的潜伏期较长时间导致电池过分拥挤样品表面，差的地形图像。表面?...

对生产设备表面和皮肤伤口生物膜存在一个问题，医疗行业作为生物膜是顽抗移走，并可能导致疾病传播和抗菌药物耐药率增加。附件是在生物膜形成的第一步，是由于它的可逆性1-3中最关键的步骤。基材表面特性发挥的微生物附着了至关重要的作用。因素，如表面硬度，孔隙度，表面粗糙度，和疏水性已被证明以进行微生物附着;然而，一些研究考察的基材表面电位（SP）对微生物粘附的作用已经做了4,5。带负电荷的表面防止苏云金芽孢杆菌的孢子附着到云母，硅和金6。变化细胞膜电位是指示改变的细胞附着和蠕动5,7的。已经观察到，电坎ogeneous表面更容易促进微生物黏附5。表征细菌的表面电位在纳米可以提供一种新的方式来理解细菌附着动力学到各种表面，从而可以在防生物结垢策略的发展提供帮助。不像用于表征细菌，如电泳光散射，ζ电位和等电点测定的电动力学的其他方法，开尔文探针力显微镜（KPFM）允许单个细胞而不是整个培养8-11的检查。想要以比较细胞 - 细胞或生物膜电特性与高准确度和精度时，这是有利的。 KPFM是原子力显微镜（AFM）的模块。 AFM是作为扫描隧道显微镜（STM）12的直接结果。利用STM首次公布的图像是由格尔德Binnig和海因里希Rorhrer于1982年12完成</suP&gt;。其发明是能够解决的原子结构由光栅扫描一个尖锐导电尖端上的导电表面中的空气。实现激发生物学家谁很快企图使用STM对DNA，蛋白质的图像干燥的样品，和病毒12高分辨率图像的含义。 STM也可以使用专门的探针针尖13的液体进行。这显示出由Lindsay 等 ，谁使用STM和AFM图像的DNA分子在10mM的HClO 4和水，在金电极13。 KPFM已被证明在确定的DNA和蛋白质分析25和生物分子与配体相互作用26的表面电位。 KPFM操作，通过测量的AFM导电悬臂刀片和一个理想的导电性样品之间的接触电势差（CPD）（ 图1，I）14,15。样品不一定必须是导电的（ 即，生物SAMPLES）。成像可以进行对云母，玻璃和硅表面（非导电），只要该非导电表面是薄且有一个基本的导电材料6,7。在CPD相当于表面电位的电压，并且可以被描述为在尖端（φ 尖 ）和样品（φ 样品 ）之间的功函数的差，通过负电子电荷除以（ - E）。当导电针尖被带到靠近一个样品表面由于在费米能量的差（ 图1，二，一 ）15（由距离 d分开），静电力（F ES）被产生。在这一点上，针尖和样品（E v）的的真空能量处于平衡和对准。在使前端接近样品表面时，尖端与样品表面接触到电接触，并作为平行平板电容器（ 图1，二B </strong&gt;的）14,15。在该点上，该尖端与样品表面的费米能量变得对准，达到稳态平衡（ 图1，二，二）。针尖与样品表面将收取和为 V CPD将形成因于 E V&#39;s和功函数的差。 A F ES作用于电接触面积，由于形成V CPD。这个力通过外部V DC到具有相同大小的形成V CPD前端应用程序随后无效（ 图1，二，三）。此DC电压施加消除表面电荷中的电接触的面积，并且V DC必要消除第V CPD在 F ES的量等于所述差在SA之间的功函数mple面和小费15。应当指出的是，尖的功函数是已知的，它是由制造商提供。在所有KPFM方法，AC电压（V AC，大约100 - 500毫伏）也被施加到尖端，以便产生所述针尖和样品14之间振荡电力。这个测量的变化以V CPD和/ 或 F ES时提供更好的分辨率。在这点上，变化的频率或电振荡的振幅可通过V DC被纠正，以及表面可以产生潜在的地图。从这些地图的特定区域的数据，可以进一步分析，以提供关于特定地形特征的电信息。 （1）提升模式，（2）调幅（AM）模式，及（3）的频率调制（FM）模式14,16:KPFM可以在三种模式下操作。升降模式为初始增量arnation KPFM的。升降机模式依赖于在其中振荡尖端在表面拖动以获得地形图像的两遍方法。用于第二遍的前端被升高的样品上方的预设距离（10 - 100 nm），而扫描背面横过相同的面积。由于此两遍方法，升降模式，相比AM-和FM KPFM，花费的时间最长为图像采集。提高尖端远离所述表面确保只有长范围 F ES被测量。此外，地形和表面电位测量之间的串扰被解耦在增加横向分辨率和灵敏度为代价的。 AM-KPFM提高通过使用双频率同时测量样品形貌和表面电位（单程扫描）14横向分辨率和灵敏度。在AM模式，悬臂机械振荡，一般为5％，低于它的第一谐振频率（f 0），并且电振荡（叔hrough一个V AC）在其第二谐振频率（f 1）。变化的f 0的引线的振幅地形数据的生成，而变化f中1的幅度时，由于改变在F中ES和 V CPD，得到表面电位的测量数据。 f 0的和f悬臂1通过显著频率和能量为信号串扰最小化14是分开的。原子力显微镜头电子箱（HEB）分离出两个信号给这两个地形和一次扫描同时表面电位的数据。 FM-KPFM提高对生物表面14的分辨率甚至比AM-KPFM。 FM-KPFM作品不同于AM-KPFM，它衡量的变化静电力梯度，而不是静电力（F ES）15。像AM模式，FM模式利用双频率和SINGLe通扫描机制获取地形，同时表面电位数据14。在FM模式中，悬臂机械振动在f 0和在低的改进的频率振荡电（F 模 ，典型的是1 - 5千赫）。经静电相互作用，女0和f MOD叠加可产生边带f 0的±˚F 国防部 。这些边带是变化的静电力非常敏感，并且可以从f 0的通过AFM的HEB分离。既然调频KPFM测量变化的静电力梯度，尖端顶点的形状和它的维护/完整性发挥整体表面电位分辨率14,15关键的作用。采用调幅表面电位分辨率和FM模式中的1纳米的范围内横向14 -16。应当指出的是KPFM成像能够在非极性液体中来实现，并且更近来，已经显示出在低离子（&lt;10毫米）极性液体进行（在开环KPFM模式不需要一个偏压反馈，从而消除的DC偏置的应用），例如milliQ水中;然而，KPFM成像尚待于活细胞中的极性溶液17-20进行。带SP显像液相关的额外的挑战是，解决方案通常用于维持细胞（ 即，磷酸盐缓冲盐水）具有高浓度的移动离子，这将导致法拉第反应，偏置感应电荷动力学，和离子扩散/再分配20。因此，对于这个实验中，测量是从干燥的和死的MRSA细胞上在环境条件下聚 - L - 赖氨酸官能不锈钢和金表面。成像可以在空气中进行或真空条件上是先前已经干燥或固定到表面20的生物样品。潮湿的条件下也被证明影响表面6的KPFM成像。 在这项研究中，我们采用FM-KPFM和AFM研究SP对耐甲氧西林金黄色葡萄球菌 USA100（MRSA）到聚-L-赖氨酸官能不锈钢和金表面的附着的作用。 MRSA最近获取了状态作为一个多药耐药（MDR）"超级病菌"，因为它的许多β内酰胺类抗生素和头孢菌素类21自然选择的阻力。 MRSA感染现在更加具有挑战性的，困难的，强大的治疗，导致使用更苛刻的抗菌剂，如万古霉素或具有在人类毒性较高的水平，因此不能被用作长期治疗22恶唑烷酮。不锈钢被选择，因为它的医疗相关性和作为金用作对比金属中的皮下注射针头，便盆，门把手，水槽等的材料共同使用。 FM-KPFM被用来研究是否微生物膜的SP时附着改变为衬底。

<table><tbody><tr><td>5500ILM Atomic Force Microscope</td><td>Agilent Technologies</td><td>#N9435S</td><td></td></tr><tr><td>AFM/STM Metal Specimen Discs</td><td>TED PELLA, INC.</td><td>#16219</td><td>Stainless steel sample discs</td></tr><tr><td>DPE (Low-Noise) Conductive SPM Probes</td><td>Mikromasch</td><td>#HQ:DPE-XSC11</td><td>There are 4 Pt-coated cantilevers per chip. We utilized cantilever B for experiments.</td></tr><tr><td>PELCO Gold Coated AFM/STM Metal Specimen Discs</td><td>TED PELLA, INC.</td><td>#16218-G</td><td>Gold sample discs</td></tr><tr><td>PicoView Software</td><td>Agilent Technologies</td><td>#N9797B</td><td>5500ILM Atomic Force Microscope imaging software</td></tr><tr><td>Pico Image Software (Pico Image Basic)</td><td>Agilent Technologies</td><td>#N9797AU-1FP</td><td>5500 ILM Atomic Force Microscope post-image processing software</td></tr><tr><td>Scilogex D3024 High Speed Micro-Centrifuge</td><td>Thomas Scientific</td><td>#91201513</td><td>Centrifuge used in cell-washing steps to separate cells (pellet) from media</td></tr><tr><td>Trypticase Soy Agar with 5% Sheep Blood</td><td>BD</td><td>#221261</td><td>Pre-made plates</td></tr><tr><td>Tryptic Soy Broth</td><td>BD</td><td>#257107</td><td>Comes as a dry powder. Instruction on how to make come on the container.</td></tr></tbody></table>

surface potential measurement of bacteria using kelvin probe force microscopy

表面电位是起着微生物到基材表面的密合性的主导作用，一个经常被忽视的物理特性。开尔文探针力显微镜（KPFM）是原子力显微镜（AFM）的模块，其测量在纳米尺度表面之间的接触电势差。 KPFM的通过AFM的组合允许同时产生表面电位和生物标本的地形图，如细菌细胞。在这里，我们采用KPFM检查表面电位对微生物的粘附性医学相关的表面的效果，如不锈钢和金。表面电位的地图显示在不同的材料基板微生物膜在表面电位差。的工序高度图形生成显示在表面电位差在衬底表面和微生物之间的边界区域。变化在细胞膜表面电位已经与变化S IN细胞代谢和蠕动。因此，KPFM表示可被用来考察微生物膜表面电位在粘结到各种基底的表面的变化的有力工具。在这项研究中，我们展示的过程来描述个人的耐甲氧西林金黄色葡萄球菌 USA100细胞的表面电位使用KPFM不锈钢和黄金。

以测量的SP使用KPFM的能力依赖于原理，这两个样品表面和悬臂刀片是导电的，以某种程度。不锈钢和黄金采取行动，而MRSA被装导电表面。 KPFM拍摄图像15的MRSA细胞的两个表面上具有512×512的分辨率，并与扫描区域范围从5×5微米至10×10微米。扫描，进行与线速度范围为0.02行/秒至0.05行/秒。因此，有512个数据点每行512线扫描采集，扫描时间范围为2.84小时至7.11小时。采集到的图像的种类可以看出，在<...

Watch this Scientific Journal Video about Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy at JoVE.com

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

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

细菌表面电位测量用开尔文探针力显微镜

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

surface-potential-measurement-bacteria-using-kelvin-probe-force

Research

JoVE Journal

Bioengineering

21.7K 次观看.  University of Guelph. Here, we present a protocol explaining the use of Kelvin probe force microscopy as a tool for generating high resolution nano-scale surface potential maps. This tool was applied to assess the role of surface potential on the binding capacity of microorganisms to substrate surfaces. 

视频: Surface Potential Measurement of Bacteria Using Kelvin Probe 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

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

细菌附着在实验室测量的植物表面

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

环境科学

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 ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

原子力显微镜与红外光谱相结合，作为探测单一细菌化学的工具

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

化学

Co-localizing Kelvin Probe Force Microscopy with Other Microscopies and Spectroscopies: Selected Applications in Corrosion Characterization of Alloys

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin probe, both of which measure the Volta potential difference (VPD) between an oscillating probe tip and a sample surface by applying a nulling voltage equal in magnitude but opposite in sign to the tip-sample potential difference. By scanning a conductive KPFM probe over a sample surface, nanoscale variations in surface topography and potential can be mapped, identifying likely anodic and cathodic regions, as well as quantifying the inherent material driving force for galvanic corrosion.
Subsequent co-localization of KPFM Volta potential maps with advanced scanning electron microscopy (SEM) techniques, including back scattered electron (BSE) images, energy dispersive spectroscopy (EDS) elemental composition maps, and electron backscattered diffraction (EBSD) inverse pole figures can provide further insight into structure-property-performance relationships. Here, the results of several studies co-localizing KPFM with SEM on a wide variety of alloys of technological interest are presented, demonstrating the utility of combining these techniques at the nanoscale to elucidate corrosion initiation and propagation.
Important points to consider and potential pitfalls to avoid in such investigations are also highlighted: in particular, probe calibration and the potential confounding effects on the measured VPDs of the testing environment and sample surface, including ambient humidity (i.e., adsorbed water), surface reactions/oxidation, and polishing debris or other contaminants. Additionally, an example is provided of co-localizing a third technique, scanning confocal Raman microscopy, to demonstrate the general applicability and utility of the co-localization method to provide further structural insight beyond that afforded by electron microscopy-based techniques.

Kelvin probe force microscopy (KPFM) measures surface topography and differences in surface potential, while scanning electron microscopy (SEM) and associated spectroscopies can elucidate surface morphology, composition, crystallinity, and crystallographic orientation. Accordingly, the co-localization of SEM with KPFM can provide insight into the effects of nanoscale composition and surface structure on corrosion.

开尔文探针力显微镜与其他显微镜和光谱学共定位:合金腐蚀表征中的选定应用

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin ...

工程学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

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

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

测量细菌细胞的抗弯刚度，使用光学陷阱

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

生物学

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record forces curves on 1000 cells in 4 hours automatically. To maintain a 4 hour analysis time, the number of force curves per cell is reduced to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, can center the tip on the first cell of the array and then move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties, etc. With the automation and the large number of cells analyzed, one can access the behavior of the cell population. This is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells

This protocol aims to automate AFM measurements on hundreds of microbial cells. First, microbes are immobilized into PDMS stamp microstructures and then force spectroscopy measurements are performed automatically on hundreds of immobilized cells.

数百个 C.白化病 细胞生物原子力显微镜测量自动化

The method presented in this paper aims to automate Bio-AFM experiments and the recording of force curves. Using this method, it is possible to record ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

凸出力显微镜: 一种量化细胞突起力的方法

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. 
AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). 
A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.

Here, we present the application of atomic force microscopy (AFM) as a simple and fast method for bacterial characterization and analyze details such as the bacterial size and shape, bacterial culture biofilms, and the activity of nanoparticles as bactericides.

接触模式原子力显微镜作为形态观察和细菌细胞损伤分析的快速技术

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the ...

细菌表面电位测量用开尔文探针力显微镜

摘要

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此视频中的章节

原子力显微镜成像的细菌固定化

细菌附着在实验室测量的植物表面

测微探针进行远程磁驱动

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

原子力显微镜与红外光谱相结合，作为探测单一细菌化学的工具

开尔文探针力显微镜与其他显微镜和光谱学共定位:合金腐蚀表征中的选定应用

测量细菌细胞的抗弯刚度，使用光学陷阱

数百个 C.白化病细胞生物原子力显微镜测量自动化

凸出力显微镜: 一种量化细胞突起力的方法

接触模式原子力显微镜作为形态观察和细菌细胞损伤分析的快速技术

细菌表面电位测量用开尔文探针力显微镜

摘要

探索更多视频

此视频中的章节

原子力显微镜成像的细菌固定化

细菌附着在实验室测量的植物表面

测微探针进行远程磁驱动

生物膜荧光探针力:受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

原子力显微镜与红外光谱相结合，作为探测单一细菌化学的工具

开尔文探针力显微镜与其他显微镜和光谱学共定位:合金腐蚀表征中的选定应用

测量细菌细胞的抗弯刚度，使用光学陷阱

数百个 C.白化病 细胞生物原子力显微镜测量自动化

凸出力显微镜: 一种量化细胞突起力的方法

接触模式原子力显微镜作为形态观察和细菌细胞损伤分析的快速技术

数百个 C.白化病细胞生物原子力显微镜测量自动化