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。

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
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			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">5500ILM Atomic Force Microscope</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9435S</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">AFM/STM Metal Specimen Discs</td>
			<td style="width:71px;">TED PELLA, INC.</td>
			<td style="width:119px;">#16219</td>
			<td>Stainless steel sample discs</td>
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			<td height="42" style="height:42px;width:216px;">DPE (Low-Noise) Conductive SPM Probes</td>
			<td style="width:71px;">Mikromasch</td>
			<td style="width:119px;">#HQ:DPE-XSC11</td>
			<td>There are 4 Pt-coated cantilevers per chip.&#160; We utilized cantilever B for experiments.</td>
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			<td height="63" style="height:63px;width:216px;">PELCO Gold Coated AFM/STM Metal Specimen Discs</td>
			<td style="width:71px;">TED PELLA, INC.</td>
			<td style="width:119px;">#16218-G</td>
			<td>Gold sample discs</td>
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			<td height="63" style="height:63px;width:216px;">PicoView Software</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9797B</td>
			<td>5500ILM Atomic Force Microscope imaging software</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pico Image Software (Pico Image Basic)</td>
			<td style="width:71px;">Agilent Technologies</td>
			<td style="width:119px;">#N9797AU-1FP</td>
			<td>5500 ILM Atomic Force Microscope post-image processing software</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scilogex D3024 High Speed Micro-Centrifuge</td>
			<td style="width:71px;">Thomas Scientific</td>
			<td style="width:119px;">#91201513</td>
			<td>Centrifuge used in cell-washing steps to separate cells (pellet) from media</td>
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			<td height="42" style="height:42px;width:216px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">#221261</td>
			<td>Pre-made plates</td>
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			<td height="21" style="height:21px;width:216px;">Tryptic Soy Broth</td>
			<td style="width:71px;">BD</td>
			<td style="width:119px;">#257107</td>
			<td>Comes as a dry powder.&#160; Instruction on how to make come on the container.</td>
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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

Measurement of Bioelectric Current with a Vibrating Probe

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 &mu;m), which can detect ionic currents in the &mu;A/cm2 range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 &mu;A/cm2. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

生物电电流测量振动探头

Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. ...

科研

生物工程

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

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 ...

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

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

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

重组DNA和蛋白质复合物，用原子力显微镜的可视化

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

生物传感器检测抗生素耐药金黄色葡萄球菌细菌

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

使用原子力显微镜测量活细胞的力学性能

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

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

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

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

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

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

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

原子力显微镜成像和支持的脂质双层力谱

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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

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

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

荧光技术结合测定活细胞中的力敏感蛋白动力学

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