这项工作是由美国国立卫生格兰特GM086822研究院资助。作者想感谢博士。亚历克Pakhomov保罗华莱士（华盛顿纳米用户设施Univ.（NTUF））和安德烈斯莱德（布鲁克AXS）的专家技术援助。 DNA原子力显微镜成像是在大学进行。华盛顿NTUF，国家纳米技术基础设施网络的成员。好心的2xGal4 2xGRE luciferease质粒是由基思山本博士（Univ.加州，旧金山）实验室提供。

1。准备的DNA和蛋白质进行成像污染物的样本<ol><li>分离生物分子成像和地方在一个合适的缓冲溶液。成像的蛋白可能纯化使用液相色谱仪 6，免疫吸附其次是 1,2抗体和沉淀，去除或Profinity确切的纯化和标记清除（ 图 2A ）。隔离的DNA分子成像小量纯化（ 图2B）。 </li><li>确认成像采用SDS - PAGE和免疫印迹的蛋白质样品的组成和纯度。对于DNA，确认序列，完成了限制性内切酶消化和琼脂糖凝胶电泳和纯洁性。 </li><li>孵育样品在原子力显微镜的吸附缓冲，促进坚持以云母样品。对于蛋白质样品，10毫米HEPES缓冲液可用于。以及可能会增加额外的低离子强度的盐或其他辅助因子。对于DNA样本，包括一个二价阳离子（如镁2 +或Ni 2 +的 ），以促进吸附在云母基片。一个合适的Mg 2 +含缓冲的DNA是10毫米的Tris，pH值7.5，10毫米氯化钠，2毫米氯化镁2 3。另一种Ni 2 +的缓冲是10毫米的HEPES，pH为6.8，10毫米NiCl 2。 </li><li>稀释样品的吸附缓冲5微克/毫升的浓度。轻轻地混合。而显微镜正准备冰商店。 </li></ol> 2。安装原子力显微镜探针<ol><li>液体细胞探头支架放置到相应的悬臂安装扩展坞。 </li><li>找到长，厚的尖锐氮化杆（SNL）探针悬臂（简称为“B”悬臂）用于成像。小心地转移到液体细胞探头支架，确保针尖保持直立。技术说明：必须格外小心支付而探头在传输过程中从任何高度下降，可能会损坏或破坏悬臂或提示。一个microlever SNL（MSNL）探头可以用来作为替代的SNL探头。 </li><li>从扩展坞中取出探头支架和光显微镜下检查，以确保它是完整的和正确地固定在液体中的细胞探头支架悬臂。 </li><li>拧紧滚花头紧固螺钉，小心地从仪器衔接大会的原子力显微镜头。技术说明：必须格外小心，同时操纵原子力显微镜头，甚至在短距离（例如，如果不正确地坐在燕尾大会）下降，可能造成实质损害。 </li><li>反转的原子力显微镜头和液体的细胞探头支架牢牢地推到了四针，在原子力显微镜头的基础。小心地返回到仪器衔接大会的原子力显微镜头。松开滚花头紧固螺钉固定到仪器原子力显微镜头。 </li></ol> 3。定位悬臂尖端，结合激光，并调整光电探测器<ol><li>使用原子力显微镜的仪器软件，找到板载光镜下的移动旋钮在悬臂的针尖。调整显微镜的照明，如果有必要提高识别提示。携带到悬臂尖端仪器软件上显示的十字线接近结束。 </li><li>光学控制器上的软件的向上和向下箭头的调整，使悬臂尖端成为关注的焦点。使用慢（S）或中等（M）的速度，同时调整光学。 </li><li>将悬臂尖端的激光利用激光调节旋钮。移动调节旋钮，直到红色激光点内的白色照明点。跟踪沿悬臂使用锯齿形图案的胳膊，直到激光定位悬臂尖端。正确对齐，会出现一个强烈的反射点的原子力显微镜头的前窗。 </li><li>中心的光电探测器，通过调整探测器上的原子力显微镜头旋钮。仪器软件的探测器显示的十字线，这将调整激光点。所观察到的信号的总和应约4-6。 </li></ol> 4。定位原子力显微镜头和调整悬臂<ol><li>将新鲜剥离的云母片磁化样品支架上的原子力显微镜的持有人板上面附着一个金属原子力显微镜标本光盘。此设置将被用于成像在第1步准备的DNA或蛋白质样品前位置的原子力显微镜头。 <ol><li>技术说明：磁化的样品架和液体细胞探头支架紧固机制和探头持有人比其他样品厚。如果目前有云母样品持有人大会和原子力显微镜头的不足之间的间隙，移动的原子力显微镜头几次选择仪器软件的退出图标。 </li></ol></li><li>旋转原子力显微镜的持有人定位是最初的成像板，使标本光盘。确保云母基板的原子力显微镜头下的中心。 </li><li>对移动的原子力显微镜头云母标本的焦点表面控制仪器的软件光盘的表面。选择在Z电机中（M）的速度，并继续对表面的原子力显微镜头，直到约2毫米以上云母。此时，转用慢（S），Z电机的转速，以避免突然之间的探头和表面（称为崩溃​​的提示）接触。 </li><li>继续移动探针接近云母基材表面，特色鲜明，直到在云母表面或尖反射重点。表面和尖端的反映，使用“的重点是：”焦平面仪器软件的图标之间切换。 </li><li>选择仪器软件的调整图标和调整的悬臂。推荐的SNL和MSNL悬臂探针的共振频率为20-60千赫。选择一个5％的峰值偏移。技术说明：充足的悬臂调整是必需的足够的样品成像。 </li></ol> 5。成像云母样品表面<ol><li>参与到云母表面的探头。设置初始扫描大小为10微米，扫描速度为1 Hz。积分增益和比例增益，可以初步设定为0.2和0.4，分别为。根据需要调整，为了跟踪和追溯扫描大致重叠收益。降低的幅度设定值将增加探头和云母之间的相互作用，并确保参与。 </li><li>捕捉10微米领域的完整的扫描图像。减少扫描尺寸为5，1，和0.5微米，捕捉每一个领域的完整图像。根据需要调整增益和扫描速度。捕获云母表面的图像提供的DNA和含蛋白质的样品进行比较的一个基准。 </li><li>放开探头单上脱离仪器软件图标点击。 </li></ol> 6。成像生物分子的利益<ol><li>轻轻地混合在第1步准备的DNA或蛋白质样品，并准备加载到成像的云母表面。混合45新鲜的吸附缓冲液（最终样品的浓度为0.5微克/毫升）5μL准备好的样品。 </li><li>小心地添加50μL的0.5微克/毫升含有生物分子溶液直接到云母表面。不移动探头或原子力显微镜头，并确保样品溶液（但不吸管尖）与探头接触。 </li><li>暂停5分钟，让样品中的生物分子坚持以云母表面。 </li><li>在5分钟后，一个额外的吸管50-100吸附缓冲液注入液相细胞探头支架结束，小心不要触摸探头，原子力显微镜头，或用吸管尖云母。这将形成一个半月板，并允许在流体中的AFM轻敲模式成像。图3显示了流体细胞，悬臂式，样品，样品架，和半月板的最后安排。 </li><li>调整光探测器，并在必要时重新调整激光的悬臂。技术说明：查看探针和激光准直是复杂的，由于加入样品缓冲探头支架引起的衍射图样。悬臂式手动重新调整，如果需要的话。 </li><li>再搞探头使用的仪器软件。调整垂直偏转，积分增益，并在必要时的比例增益。这将开始重新成像的过程中，云母节前夕加样和捕捉图像扫描。 </li><li>增加扫描尺寸（500 NM - 10微米），以确定感兴趣区域。变焦和0.5 Hz的频率降低扫描速度，以提高图像分辨率</li><li>一旦成像完成后，脱离探针仪器上的软件选择退出图标几次。确保足够的间隙之间的原子力显微镜头和液体细胞或标本盘拆装前的标本光盘。 </li><li>用蒸馏水彻底冲洗液细胞的探头支架和标本光盘，以防止残留的吸附缓冲蒸发形成的盐晶体。用压缩空气干燥。 </li></ol> 7。代表性的成果： 图4中的原子力显微镜图像的例子。云母基片（一）提供了一个分子平坦的表面上的DNA和蛋白质可以吸附。前生物分子样品的成像云母提供了一个消极的成像噪声的控制和评估。它还提供了一个悬臂适当的调整和随后的样品成像会取得成功的保证水平。双链质粒DNA（B，D），推定超，很容易确定其不对称的外观和以前不起眼的云母基板上沉积的统一。谨慎的粒径（C，E的）的蛋白复合物，也从云母基板唯一区别的。粒度的差异表明样本异质性，可有用的逼近蛋白复合物的化学计量学或生化活性。一般的对角线形状和亲一致的方向在C组中观察到的蛋白颗粒是一种成像神器，如蛋白质会预计在云母基板上以随机方式面向。所观察到的神器可能的原因是一个物理的提示异常或原子力显微镜成像在过于快速的扫描速率。虽然提示卷积（ 如图1B所示）防止绝对长度测量计算，在X和Y轴（Z轴）高度测量和相对的X和Y的测量，可还是估计的成像生物分子的生物物理特性作出了有益的。 <img src="/files/ftp_upload/3061/3061fig1.jpg" alt="图1"/> 图1：轻敲模式原子力显微镜（AFM）的原理介绍。一，在显微镜。在悬臂年底是一个尖锐的的提示，振荡向上和向下，因为它在云母基板的表面进行扫描，生物分子复合体吸附。由于扫描仪在X和Y方向移动，激光束反射到一个位置敏感的光电二极管探测器地图提示动作，因为它传递坐在上的生物分子复合物，垂直（Z）距离，从后面的悬臂云母。 B.在提示卷积造成的X和Y方向的图像失真。传统的氮化硅针尖的名义半径大于颗粒成像，针尖接触样品的边缘，因为它穿越表面。在影像出现在X和Y方向的大颗粒，但没有Z轴方向的提示卷积结果。提示使用一个较小的名义刀尖半径的，可以减少这种效应。 <img src="/files/ftp_upload/3061/3061fig2.jpg" alt="图2"/> 图2：immunoadsorbed蛋白质和DNA样品制备的插图。一，发行immunoadsorbed的GR•HSP70蛋白的单克隆抗体（mAb）蛋白A琼脂糖凝胶（PAS）的颗粒复杂。孵化与含有单克隆抗体的抗原表位肽的immunopellet，将有利于颗粒GR•HSP70蛋白复合物的释放，使复杂的收集上清，可视化的AFM。乙，DNA的制备，原子力显微镜成像，需要使用一个二价阳离子吸附缓冲。二价阳离子增加云母基板上的DNA的亲和力。 <img src="/files/ftp_upload/3061/3061fig3.jpg" alt="图3"/> 图3：最后的安排的原子力显微镜头，液体细胞探头支架，样品，标本持有人，和缓冲区半月板。一，护理必须采取沉积到云母基板时，样品和额外的缓冲区，以避免吸管尖和原子力显微镜头，液体通话，和云母基板之间的身体接触。 B，A的放大倍率从标本光盘缓冲区半月板跨度液体细胞探头支架。 <img src="/files/ftp_upload/3061/3061fig4.jpg" alt="图4"/> 图4：云母基板的原子力显微（一），2xGal4 - 2xGRE luciferease质粒DNA（B，D），和GR•HSP70蛋白复合物在缓冲溶液中（C，E的） 。云母，与DNA和蛋白质的可视化控制图像。遗传资源•HSP70蛋白复合物的制备与HSP70和抗体珠颗粒释放使用一个单克隆抗体竞争性抗体（如图2A所示），然后催芽遗传免疫吸附。 DNA的制备按常规质粒小量。面板A，B和C相当于放大倍率（比例尺= 200 nm）的一样，都是面板D和E（比例尺= 40纳米）。

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AFM成像个别无涂层的生物分子在水溶液和近生理缓冲的实时解决方案的能力提供了一个独特的微观技术。这使得个别蛋白质和DNA分子，以及多蛋白复合物和蛋白质- DNA复合物的可视化。与原子力显微镜成像大分子颗粒，可用于评估样品的均匀性，以确定所观察到的粒子组成部分的物理安排的有用。这种方法观察单个大分子颗粒可以成为一个有用的辅助手段，传统的生化技术，如免疫检测，聚丙烯酰胺凝胶电泳，尺寸排阻柱色谱，提供重要的总结性的数据，代表10 3 -10 11个人利益的生物分子复合物目前，一个样本。 对多蛋白复杂的化学计量学，生物分子相互作用，和辅酶要求的研究都可以用原子力显微镜研究。这里介绍的方法可以调整，以适应具体的生物物理问题的兴趣。例如，使用液体电池探头支架能够交换缓冲区，它可以测定蛋白质复合物的形成辅助因子的要求。近实时的，甚至可以形成和DNA -蛋白质装配分离，而成像。利益的特定序列（如一个推定响应元素或新的蛋白质结合序列）与虚拟结合蛋白的混合，产生的DNA可与，和成像，分子间的相互作用提供直接证据。 轻敲模式原子力显微镜成像是大大降低复杂，如果干的样品，而不是在水溶液中的样本，和线性DNA站封闭圈DNA质粒都被这种方式3,9成像利用。这里介绍的方法是利用缓冲解决方案的样本，以提供更多的生理成像环境。其他值得一提的成像蛋白质的方法也应予以考虑，包括近场红外显微镜 10 。免疫吸附原子力显微镜驸互补提供了一个机会，准备无数的蛋白质复合物，使用以及建立的生化技术，然后可视化immunoadsorbing抗体珠颗粒释放。例如，immunoadsorbed遗传资源已检测的关联蛋白的分子伴侣HSP70 1以及动力蛋白马达蛋白2，使用这种方法，以估计复杂的大小和化学计量。粒度和生物物理特性（如刚性）已确定在原子力显微镜成像样品11,12中观察到的生物分子的身份。它也可以确认生物大分子相互作用的生物分子（如配体或单克隆抗体），将绑定到其目标，并造成颗粒大小的增加，如果目标生物分子是目前除了的身份。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>外形尺寸3100 </td><td>布鲁克</td><td>外形尺寸3100 </td></tr><tr><td>尺寸液电池</td><td>布鲁克</td><td> DTFML - DD - </td></tr><tr><td>夏普氮化杆（SNL）氮化硅AFM探针</td><td>布鲁克</td><td> SNL - 10 </td></tr><tr><td>氮化Microlever急剧杆（MSNL）氮化硅AFM探针</td><td>布鲁克</td><td> MSNL - 10 </td></tr><tr><td>奈米级的AFM仪器软件</td><td>布鲁克</td><td> 004-132-000 </td></tr><tr><td>金属原子力显微镜标本光盘</td><td>特德佩拉</td><td> 16208 </td></tr><tr><td>级V1云母光盘，12毫米</td><td>特德佩拉</td><td> 50-12 </td></tr></tbody></table>

visualization of recombinant dna and protein complexes using atomic force microscopy

原子力显微镜（AFM）允许个别蛋白质，DNA分子，蛋白质 - 蛋白质复合物，和DNA -蛋白质复合物的可视化。在显微镜的悬臂是纳米级的探针，它遍历图像领域从纳米到微米，测量海拔在任何给定的的点在基片表面上的大分子。静电作用引起的蛋白质，脂类，和核酸，以松散的随机取向的衬底的重视，并允许成像。生成的数据类似地形图，其中大分子三维颗粒离散大小（ 图1）1,2解决。轻敲模式原子力显微镜涉及的悬臂的反复振荡，允许相对较软的生物材料如DNA和蛋白质的成像。原子力显微镜等纳米显微技术显着的好处之一是其相对的适应性，在水溶液中的缓冲区，其中包括近生理缓冲条件，实时，无染色或涂层样品成像，可视化单个蛋白和大分子复合物。 这里介绍的方法，介绍了成像的DNA和immunoadsorbed转录因子（即糖皮质激素受体 ​​，GR）的缓冲溶液（ 图2） 。 Immunoadsorbed蛋白质和蛋白质复合物，可以分开immunoadsorbing抗体珠颗粒与抗体的抗原表位，然后成像（ 图2A）竞争。这使得前成像利益的生物分子的生化处理。一旦纯化，DNA和蛋白质，可以混合，可以成像，以及由此产生的相互作用的复杂。 DNA的云母结合需要二价阳离子如镍2，3 +或Mg 2 +的 ，这可以被添加到样本缓冲区保持蛋白的活性。原子力显微镜已经使用了类似的方法，利用可视个人的酶，包括RNA聚合酶和修复酶5，约束个人的DNA链。这些实验提供的蛋白质 - 蛋白质和DNA -蛋白质生物物理相互作用在分子水平上发生显着的洞察力。原子力显微镜成像个别大分子颗粒，可确定粒子的同质性，并有助于确定的成像颗粒组成部分的物理安排。虽然目前GR伴侣蛋白复合物1,2和DNA链的GR可以结合可视化开发方法，它可广泛应用于成像DNA和蛋白质样品从多种来源。

Watch this Scientific Journal Video about Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy at JoVE.com

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

是描述一个轻敲模式原子力显微镜（AFM）的可视化的质粒DNA，细胞质的蛋白质和DNA -蛋白质复合物的方法。该方法包括准备样品的原子力显微镜成像，生化操纵的替代方法。在近生理缓冲条件下观察DNA含有特定的蛋白相互作用的区域。

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 ...

visualization-recombinant-dna-protein-complexes-using-atomic-force

Research

JoVE Journal

Bioengineering

22.3K 次观看.  Seattle University. 是描述一个轻敲模式原子力显微镜（AFM）的可视化的质粒DNA，细胞质的蛋白质和DNA -蛋白质复合物的方法。该方法包括准备样品的原子力显微镜成像，生化操纵的替代方法。在近生理缓冲条件下观察DNA含有特定的蛋白相互作用的区域。

视频: Visualization of Recombinant DNA and Protein Complexes Using Atomic 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

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

量化的内皮细胞糖复合物的力学性能，用原子力显微镜

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

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

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

使用原子力显微镜测量活细胞的力学性能

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

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. Kelvin probe force microscopy (KPFM) is a module of atomic force microscopy (AFM) that measures the contact potential difference between surfaces at the nano-scale. The combination of KPFM with AFM allows for the simultaneous generation of surface potential and topographical maps of biological samples such as bacterial cells. Here, we employ KPFM to examine the effects of surface potential on microbial adhesion to medically relevant surfaces such as stainless steel and gold. Surface potential maps revealed differences in surface potential for microbial membranes on different material substrates. A step-height graph was generated to show the difference in surface potential at a boundary area between the substrate surface and microorganisms. Changes in cellular membrane surface potential have been linked with changes in cellular metabolism and motility. Therefore, KPFM represents a powerful tool that can be utilized to examine the changes of microbial membrane surface potential upon adhesion to various substrate surfaces. In this study, we demonstrate the procedure to characterize the surface potential of individual methicillin-resistant Staphylococcus aureus USA100 cells on stainless steel and gold using KPFM.

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. ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

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 ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

枯草芽孢杆菌中细菌体和内膜的可视化

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

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 ...