这项研究是，美国能源部生物和环境研究办公室，并授予来自弗吉尼亚州的联邦卫生研究委员会的资金赞助。美国能源部橡树岭国家实验室管理有限责任公司，UT斯达康，巴特尔，根据合同号DE - AC05 - 00OR22725。

1。云母的准备： <ol><li>削减云母的大小要适合AFM显微镜（约22 × 30毫米）的剪刀（电子显微镜学）。 </li><li>顺劈斩双方云母，一般用胶带去除的外层，直到顺利不间断层保持。 </li></ol> 2。明胶溶液的制备： <ol><li>实验室瓶100毫升的蒸馏水。 </li><li>一瓶在微波加热，直到水开始沸腾。 （如果不可用微波炉，水可以在一个热点板块的烧杯加热。） </li><li>称取0.5克，明胶（Sigma公司的G - 6144 G - 2625或G - 2500），加入热蒸馏水。 </li><li>轻轻摇动瓶子直到明胶完全溶解。 </li><li>明胶溶液冷却到60-70 ° C。 </li><li>削减云母可斜射到小烧杯中（20毫升）倒入约15毫升。 </li></ol> 3。云母涂层： <ol><li>云母广场淹没到温暖的明胶溶液，并迅速撤出，云母涂层（图1a）。 </li><li>涂装后，立即支持云母，边缘，在纸巾上，周围空气中的干（图1b）。明胶包覆云母可用于至少2周。多余的明胶溶液可冷藏保存，使用约一个月只需再加热原液之间60-70 ° C。必须小心，以确保明胶期间未再加热煮沸。 </li></ol> 4。安装明胶包覆云母的细菌： <ol><li>佩莱1毫升细菌培养在800至4​​500 RCF（0.5 - 1.0在600 nm的OD）。根据上的细菌，使用最低的区域合作框架，将沉淀的细胞〜5分钟。 </li><li>在相同体积的过滤去离子水或缓冲液洗涤沉淀。 </li><li>再通过离心收集细胞。 </li><li>及时nanopure去离子水或缓冲液500μL重悬沉淀。 （菌悬液应明显混浊才能有足够的浓度细胞的原子力显微镜成像。） </li><li>应用细胞悬液（10-20μL）明胶包覆云母和表面上的传播，使用吸头（图1c）。应采取不接触枪头明胶的表面，而申请或传播细菌的解决方案。 </li><li>允许休息10分钟，并与流的水或缓冲液（图1 CD）冲洗表面。整个过程中，照顾，以确保该样本是不允许干的。 </li></ol> 5。代表性的成果： 图2显示了已安装在明胶包云母和原子力显微镜成像的细菌细胞的例子图像。在我们的实验室，样品通常使用一个PicoPlus原子力显微镜（安捷伦科技，寺，AZ），配备一个100微米的扫描头成像。该仪器操作在0.5-1.0行/秒的扫描速度在128-512像素，每行扫描通常使用的悬臂Veeco的氮化硅探针与标称弹簧常数的范围从0.01至0.1 NN /纳米（MLCT AUHW，Veeco公司，圣巴巴拉分校，加利福尼亚州）。 <img src="/files/ftp_upload/2880/2880fig1.jpg" alt="图1"/> 图1，在温度60-70℃放置在20毫升的烧杯中明胶。使用镊子新鲜剥离的云母广场完全沉浸到明胶（一）撤销，而直立靠在一个微型离心机机架（二）一纸毛巾放在。细菌样品液滴放置在X和Y方向（C）吸管尖云母和蔓延。使样品在云母表面孵育至少10分钟后，冲洗样品在蒸馏水流。如果固定工作，你将能够看到一个不透明的点上的云母，在右侧面板（D）所示。如果固定没有工作，你会得到明确的云母片，在左侧面板。云母鳞片白斑液滴的细菌浓度，明胶处理的云母。 <img src="/files/ftp_upload/2880/2880fig2.jpg" alt="图2"/> 图2 E 的 AFM图像。 大肠杆菌 。面板（A），细胞安装在明胶包云母和0.005 M PBS成像。为了便于比较，明胶包覆云母和安装在空气中成像的细菌细胞的图像显示面板（B）。虽然隔的液体，更高的分辨率菌毛（插图面板（二））表示，在采集到的图像中清楚可见，可在空气中获得。

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没有利益冲突的声明。

许多因素可能会影响微生物细胞固定的原子力显微镜和成像。明胶，用于涂料，云母是重要的。商业明胶是孤立的，从脊椎动物，包括鱼，牛，猪的数量。的起源和处理方法，确定为细菌固定化明胶的适用性。许多来源和类型的明胶固定细菌的有效性进行评估[1]。两个最有效的明胶被发现SIGMA的G - 6144和G - 2625。从猪（猪），这些明胶被认为是低布鲁姆和中等布鲁姆分别。明胶处理过的基材和特定?...

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>明胶</td><td> Sigma公司，圣路易斯，密苏里州</td><td> G6144，G2625或G2500 </td></tr><tr><td> PicoPlus原子力显微镜</td><td>安捷伦科技公司，坦佩，AZ </td><td></td></tr><tr><td>原子力显微镜的悬臂</td><td> Veeco公司，圣巴巴拉，CA </td><td> MLCT - AUHW </td></tr></tbody></table>

bacterial immobilization for imaging by atomic force microscopy

AFM是一个高分辨率（纳米尺度）的成像工具，机械探头表面。它具有图像细胞和生物分子的能力，在液体环境中，无需化学处理的样品。为了实现这一目标，必须充分坚持样品安装表面，以防止去除扫描原子力显微镜悬臂的针尖施加的力量。在许多情况下，成功的成像取决于样品固定在安装表面。理想情况下，固定应微创样品等的代谢过程和功能属性不受损害。猪（猪）明胶涂层新鲜剥离的云母表面带负电荷的细菌可以固定在表面上，并在液体中原子力显微镜成像。明胶包覆云母上的细菌细胞固定化是最有可能是由于带负电荷的细菌和带正电荷的明胶之间的静电相互作用。有几个因素可以干扰细菌固定化，包括在其中的细菌悬浮液，明胶包覆云母，菌株和成像在其中的细菌培养基的表面特性的细菌培养时间化学成分。总体而言，使用明胶包覆云母成像微生物细胞被普遍适用。

Watch this Scientific Journal Video about Bacterial Immobilization for Imaging by Atomic Force Microscopy at JoVE.com

Bacterial Immobilization for Imaging by Atomic Force Microscopy

现场革兰氏阴性和革兰氏阳性菌，可固定在明胶包覆云母和利用原子力显微镜（AFM）在液体中成像。

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 ...

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17.2K 次观看.  Oak Ridge National Laboratory. 现场革兰氏阴性和革兰氏阳性菌，可固定在明胶包覆云母和利用原子力显微镜（AFM）在液体中成像。