Name: peering at brain polysomes with atomic force microscopy
Uploaded: 2016-03-16T15:00:00.000+00:00
Duration: 8 min 49 s
Description: Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.

用于获取小鼠组织中的做法是由身体特伦托大学的动物保护（OPBA）（意大利）批准，协议没有。 04-2015，每art.31因为法令没有。二千〇一十四分之二十六。所有小鼠均保持在特伦托大学中心综合生物学（CIBIO），意大利的模式生物设施。 注意:为避免样品的任何RNA的降解，用准备DEPC处理过的水中所有的缓冲区最大限度地减少RNase污染。 1.全脑多核​​糖体的制备<ol><li>收集脑组织（15分钟） <ol><li>安乐死用CO 2窒息野生型小鼠品系C57BL / 6 5分钟36。小心从头骨36解剖大脑出来，所述组织置于1.5ml试管，并立即将其放置在液氮中。储存在-80℃直到使用。 </li></ol></li><li>裂解物的制备（30分钟） <ol><li>使用粉碎全脑组织液氮下用研钵和研杵。 </li><li>转移约25mg粉末的冷的离心管，并立即通过快速上下吹打25倍（以避免粉碎组织的解冻）添加0.8毫升裂解缓冲液（ 见表1）的和破坏细胞。 </li><li>离心管以12,000rpm xg离心1分钟，在4℃以沉淀细胞碎片。 </li><li>将上清转移到新的微量离心管中，并保持该管在冰上15分钟。 </li><li>离心管以12,000rpm xg离心5分钟，在4℃以沉淀细胞核和线粒体。 </li><li>将上清转移到新的离心管中。 </li><li>储存在-80℃的上清液为最多6个月或立即使用它。 </li></ol></li><li>蔗糖梯度制备和离心（2小时，30分钟） <ol><li>用无RNase水（焦碳酸二乙酯处理的水（用DEPC水）或商业）洗涤超速离心管广泛的ð3％H 2 O 2 / DEPC H 2 O解决方案。 </li><li>置于冰上的管中并在每个管（参见，蔗糖溶液表1）的底部添加5.5冷的50％的蔗糖溶液。小心逐滴添加15％的蔗糖溶液滴，力求贴近相间，以便保持一个尖锐相间，直到管被完全填充。当管被完全填充，用橡胶塞将其关闭，以避免气泡形成。 </li><li>在寒冷的房间轻轻水平放下管子，并保持在此位置2小时。在此时间后，慢慢伸直管放回竖直位置，并把它们在冰上。梯度现在准备好被使用。可替代地，使用常规的梯度前制备15-50％的蔗糖梯度。 </li><li>在寒冷的房间从渐变的顶部取出仔细1.0毫升和覆盖一滴蔗糖下降与胞浆裂解液（即 STE得到的上清液p 1.2）。 </li><li>小心地将管放入水平转头的水桶。使用超速离心机离心100分钟梯度180,000 xg离心在4℃下。 </li><li>离心分离后，离开管子自己水桶20分钟，4℃，让梯度稳定。 </li></ol></li><li>蔗糖梯度分馏（2小时） <ol><li>小心地从超速离心机转子删除一个超速离心管并将其安装密度梯度分馏系统的集热设备。收集1毫升级分监测吸光度在用UV / VIS检测器（参见图1上图 ）260纳米。置于冰上收集到的馏分。 </li><li>制备30-40微升感兴趣的级分的等分试样，在-80℃，直到使用它们存储之前保持他们在冰上。不使用行多于两个冻融循环等份或蔗糖级分（ 见图1中的下图 ）。 </li></ol> &lt;/ LI&gt; </ol> 2.样品制备原子力显微镜（3小时） <ol><li>使用磁带，剥去云母片。 </li><li>用DEPC水冲洗云母片3-4次，并把它变成一个小的培养皿中。然后用空气干燥的表面上。 </li><li>覆盖用200μl1mM的硫酸镍4的云母并在室温下孵育3分钟。 </li><li>除去镍溶液中，然后用空气干燥的表面上。通过将培养皿在冰上云母进行在4℃以后所有的步骤。 </li><li>在冰上解冻1.4.2获得等份，轻轻用在云母滴添加所有样品下降。使用100-200微升尖，传播云母的整个表面上的样品。在冰上孵育样品3分钟。 </li><li>覆盖一滴云母片下降用200μl冷缓冲区的AFM（ 见表1）和孵育在冰上1小时。 </li><li>成像液<ol><li>仔细缓冲区AFM捞出洗净云母片3-4时s的200微升冷缓冲区AFM以去除多余的蔗糖。然后洗云母片用冷水洗涤液的3倍（ 见表1），同时保留的解决方案的一些微升覆盖的云母表面。 </li><li>进入3点（图像采集）。 </li></ol></li><li>成像在空气中<ol><li>小心地取出缓冲区AFM和用200μl冷缓冲区AFM洗云母3-4次以除去过量的蔗糖。然后，洗云母用冷洗涤溶液3倍（ 见表1），并使用纸排出多余的水。 </li><li>保留样品的化学罩与培养皿部分开放的顶部下干燥。 2小时后，收于RT培养皿和存储。测量样品后2-3小时，因为它们是稳定多年。 </li></ol></li></ol> 3.图像采集（热稳定后，每幅影像15分钟） 注意:固定在云母多核糖体可在空气中或在液体中进行成像，使用AC模式。 <ol><li>附加云母使用双面胶带的样本保持器。 </li><li>插入以下制造商的指示在AFM中阶段的样品架。当显像液中，如果可能的话，尝试在温度低于25℃以保持样品，以增加在时间上多核糖体的稳定性。 </li><li>选择适用于交流成像的悬臂，它安装在刀片保持器下面的制造商的指示。在这里，使用悬臂用武力2-20牛顿/米的空中成像和液体成像约为0.1牛顿/米之间不变。 </li><li>调整在悬臂激光点和零的象限检测器的信号。 </li><li>选择一个适当的驱动频率并且具有10-20纳米的振幅驱动该悬臂。 </li><li>接近样品直至前端的表面接合。 </li><li>选择2×2微米的扫描区域，至少获得512×512像素的图像（像素宽度&lt;4海里），选择一个实时背景减除模式和20-25纳米的Z比例。 </li><li>检查ŧ他图像寻找在范围25-30纳米的空气和25时30纳米液体和宽度取得时特征10和15毫微米之间的高度圆形物体的存在。调整设定值和反馈参数，直到尖锐物体可视化。背景应显示良好的样品中的相对平坦的，具有一定的2-4纳米的高度的物体（参见图2A和B）。 </li><li>如果图像看起来不错（如点3.8所示），收购在不同的采样区域的几个（至少十）的2x2微米扫描。 </li><li> （可选）。如果有必要，获取选定多聚核糖体的高分辨率的图像（ 见图2C）。 </li><li>应用软件更正（平面减法和行由行校正算法）来纠正AFM图像去除任意倾斜和漂移的影响。 </li></ol> 4.数据分析（每幅影像30分钟） <ol><li> preferent:在ImageJ的（应用比例因子可选）导出图像ially使用无损压缩格式， 例如，在TIFF格式（ 见图3A）。 </li><li>使用ImageJ的宏工具集RiboPick.ijm（在ImageJ的宏/工具集子目录被复制）来计算在多核糖体的核糖体（参见图3B），并计算样品的统计特性（ 见图3C）。 <ol><li>启动加载使用&lt;读取图像&gt;工具，初始化程序的图像。 </li><li>挑选的标准ImageJ的&lt;点选择&gt;工具（Shift +左键点击允许核糖体的多选）核糖体中心。马克与&lt;马克核糖体&gt;工具选择的核糖体。核糖体坐标将出现在自定义文本窗口。 </li><li>添加更多的核糖体为相同的多核糖体重复在点4.2.2所示的过程。 </li><li>删除使用&lt;撤消最后挑&gt;工具（开始从最后添加的核糖体取出到第一个错误标记的核糖体212;在当前的多核糖体只）。 </li><li>当完成多聚核糖体，使用&lt;新建多聚核糖体&gt;工具。作为多核糖号添加作为覆盖到图像，文本窗口被更新。 </li><li>使用&lt;减结果，并靠近&gt;写核糖体坐标文件（用于图像的默认单位），并且总结了拾取核糖体和多核糖PNG图像。原始图像被关闭没有被保存。 </li></ol></li></ol>

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Biochem. 394, 275-280 (2009).</li><li>Bernab&#242;, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+translational+control+in+non-model+stressed+organisms+by+polysomal+profiling.">Studying translational control in non-model stressed organisms by polysomal profiling.</a> J. Insect Physiol. 76, 30-35 (2015).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IH+Image+to+ImageJ:+25+years+of+image+analysis.">IH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Lauria, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RiboAbacus:+a+model+trained+on+polyribosome+images+predicts+ribosome+density+and+translational+efficiency+from+mammalian+transcriptomes.">RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes.</a> Nucleic Acids Res. , (2015).</li><li>Al Omran, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+the+Ependymal+Cilia+in+the+Lateral+Ventricles+of+the+Mouse+Brain.">Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain.</a> J Vis Exp. (100), e52853 (2015).</li><li>Warner, J. R., Rich, A., Hall, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscope+studies+of+ribosomal+clusters+synthesizing+hemoglobin.">Electron microscope studies of ribosomal clusters synthesizing hemoglobin.</a> Science. 138, 1399-1403 (1962).</li><li>Yazaki, K., Yoshida, T., Wakiyama, M., Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysomes+of+eukaryotic+cells+observed+by+electron+microscopy.">Polysomes of eukaryotic cells observed by electron microscopy.</a> J. Electron Microsc (Tokyo). 49, 663-668 (2000).</li></ol>

The authors have no competing financial interests in this paper and nothing to disclose.

由于DNA的结构被证明是至关重要来形容转录和染色质组织提高了我们的基因表达的转录调控认识的过程，它分析多聚核糖体的组织和结构，提高真实的理解是必不可少的翻译及其调控。 所描述的协议中，轻轻地固定在一个平面上的多核糖体的最佳密度，适于原子力显微镜成像被获得。使用具有方便制备的样品这些条件下，每小时近50多核糖体可以被收购，准备作进一步的分析...

蛋白质的合成是在细胞1,2-最耗能的过程。因此，并不奇怪，蛋白质丰度主要控制在翻译，而不是转录水平3,4,5。多核糖体是转换的mRNA信息为功能蛋白质读数基波高分子组分。多聚核糖体是迄今公认的大分子复合物，几个平移控制收敛6-13。尽管数百对核糖体结构14-16研究，详细的分子见解翻译的动态和多聚核糖体的拓扑结构遇到了兴趣有限。因此，原生多聚核糖核蛋白复杂的组织结构及其在翻译的潜在影响仍然是相当模糊的问题。多聚核糖体可能隐藏还是未知数有序和职能部门，潜在的镜像什么核小体和后生CONTROLS已经代表了转录领域。事实上，这样一个有趣的假设的调查需要更多的研究和新的技术途径。在此行中，结构的技术和原子力显微镜可以卓有成效协作解开的新机制，用于控制基因表达的，类似于什么是为核小体17,18来实现的。 由于核糖体14-16的发现，它的结构已被广泛表征在原核生物19,20，酵母21以及最近在人22，在蛋白质合成的基础上提供的机制的分子描述。多核糖体进行初始确认在内质网的膜，从而形成典型的2D几何机构14。正如前面提到的，多聚核糖体装配一直没有固定利率，作为核糖体结构的对象。在过去，多聚核糖体已基本上由TR研究ansmission EM为基础的技术。只是在最近，冷冻电镜技术，使纯化多聚核糖体的三维重建体外翻译系统23-26，人类细胞裂解液27或28的细胞。这些技术提供了有关在多核糖体23，26-28和在小麦胚芽多核糖体24相邻的核糖体的接触表面进行初步分子描述的核糖体的核糖体组织更精致的信息。因此，冷冻电镜断层扫描允许与分子细节核糖体的核糖体相互作用的公开内容，但它是由大量的后处理的负担，并重建分析需要大量的计算资源进行数据处理。此外，为了得到核糖体的核糖体相互作用的分子细节，核糖体的高度解决地图是必需的，并且这种参考核糖体仅可用于几种。重要的是，冷冻电镜断层扫描是无法检测无RNA。 Theref矿石，需要新的技术来全面了解机器翻译的组织。 旁冷冻电镜，原子力显微镜也已在低等真核生物29-33和人类27用作用于多核糖体的直接成像的有用工具。相比EM，AFM不需要样品固定或标签。此外，测量结果可以在接近生理条件，并具有明确确定两个核糖体和裸RNA链27上的唯一的可能性来进行。能够相对快速地执行单个多核糖体的成像，在纳米分辨率的小的后处理工作获得成千上万的图像相比，广泛和重后处理和重建分析通过冷冻电镜显微术所需的。因此，AFM数据处理和分析，也不需要昂贵的工作站和高计算能力。因此，该技术收集多核糖体的形状信息，形态特征（如高度，长度和宽度），核糖体密度的无RNA的存在和每多核糖体27的核糖体比冷冻电镜更高的吞吐量的数量。在这样的方式，AFM代表了一个强大的和互补的方式来EM技术来描绘核糖27。 在这里，我们提出了一个完整的管道从净化到AFM应用到图像和分析小鼠大脑多聚核糖体的数据分析。该协议的重点是净化问题上用于原子力显微镜成像云母基材多聚核糖体的精确沉积。除了 ​​可与通过AFM社区使用的通用软件容易地进行常规颗粒分析，一个ImageJ的34插件 ​​，称为RiboPick，提出用于计数每多核糖体27，35的核糖体的数目。

<table><tbody><tr><td>Cycloheximide</td><td>Sigma</td><td>01810</td><td>Prepararation of lysate</td></tr><tr><td>DNAseI</td><td>Thermo Scientific</td><td>89836</td><td>Prepararation of lysate</td></tr><tr><td>RiboLock RNAse Inhibitor</td><td>Life technologies</td><td>EO-0381</td><td>Prepararation of lysate</td></tr><tr><td>DEPC</td><td>Sigma</td><td>40718</td><td>Prepararation of lysate</td></tr><tr><td>Triton X100</td><td>Sigma</td><td>T8532</td><td>Prepararation of lysate</td></tr><tr><td>DTT</td><td>Sigma</td><td>43815</td><td>Prepararation of lysate</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma</td><td>D6750</td><td>Prepararation of lysate</td></tr><tr><td>Microcentrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5417R</td><td>Prepararation of lysate</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S5016</td><td>Sucrose gradient preparation</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima LE-80K</td><td>Sucrose gradient centrifugation</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Beckman Coulter</td><td>SW 41 Ti</td><td>Sucrose gradient centrifugation</td></tr><tr><td>Polyallomer tube</td><td>Beckman Coulter</td><td>331372</td><td>Sucrose gradient centrifugation</td></tr><tr><td>Density Gradient Fractionation System&amp;nbsp;</td><td>Teledyne Isco</td><td>67-9000-176</td><td>Sucrose gradient fractionation</td></tr><tr><td>AFM</td><td>Asylum Research</td><td>Cypher</td><td>Polysome visualization</td></tr></tbody></table>

peering at brain polysomes with atomic force microscopy

平移机械， 即核糖体或多聚核糖体是细胞中最大和最复杂的细胞质机械之一。多核糖体，核糖体，mRNA的，几种蛋白质和非编码RNA形成的，代表集成，其中平移控制发生平台。然而，尽管核糖体已被广泛研究，多核糖的组织仍然缺乏全面的了解。因而很多努力，需要以阐明多核糖体组织和可嵌入的翻译控制的任何新颖的机制。原子力显微镜（AFM）是一种类型的扫描探针显微镜的，允许在纳米级的分辨率采集3D图像。相比电子显微镜（EM）技术，原子力显微镜的主要优点之一是，它能够获得几千无论在空气中和溶液的图像，使该样本被接近生理条件下维持，而无需任何染色和固定的亲cedures。这里，对从鼠脑和它们对云母基板沉积多核糖的准确纯化的详细协议进行说明。该协议允许在空气和液体多核糖成像AFM和它们重建为三维物体。为了补充低温电子显微镜（冷冻电镜），该方法可以方便地用于系统的分析多核糖体和研究他们的组织。

整个小鼠大脑的蔗糖梯度多聚核糖分析 有可能从由多核糖体分析，其中根据它们的重量及尺寸分离大分子细胞裂解物纯化核糖。与多核糖体分析，从培养的细胞或组织，如在本实施例获得的细胞质裂解物，装载到一个线性蔗糖梯度和加工通过超速离心根据其沉降系数以分开40S和60S亚基，80S核糖体和多核糖体游离?...

Watch this Scientific Journal Video about Peering at Brain Polysomes with Atomic Force Microscopy at JoVE.com

Peering at Brain Polysomes with Atomic Force Microscopy

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

在脑核糖凝视与原子力显微镜

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

peering-at-brain-polysomes-with-atomic-force-microscopy

Research

JoVE Journal

Neuroscience

8.3K 次观看.  Fondazione Bruno Kessler. While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid. 

视频: Peering at Brain Polysomes with Atomic Force Microscopy

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 ...

科研

生物工程

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

工程学

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

生物化学

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, ...

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

We present a method for achieving sub-nanometer resolution images with amplitude-modulation (tapping mode) atomic force microscopy in liquid. The method is demonstrated on commercial atomic force microscopes. We explain the rationale behind our choices of parameters and suggest strategies for resolution optimization.

亚纳米级分辨率成像与调幅原子力显微镜在液体

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

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 ...

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 ...

Static Atomic Force Microscopy to Visualize and Characterize Assembled Nucleosomes

Source: Stormberg, T., et al. <a href="https://app.jove.com/v/58820">Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging.</a> J. Vis. Exp.(2019)
In this video, we demonstrate static atomic force microscopy to visualize assembled nucleosomes. This high-resolution imaging technique allows the study of the nucleosome structure.

静态原子力显微镜，用于可视化和表征组装的核小体

Source: Stormberg, T., et al. Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp.(2019)
In this video, ...

JoVE Encyclopedia of Experiments

Biological Techniques

Microscopy Techniques

Atomic Force Microscopy

Atomic force microscopy (AFM) is a type of scanning probe microscopy that can analyze topographic details of various specimens like ceramics, glass, polymers, and biological samples. AFM offers over 1000 times more resolution than the optical imaging system. Images generated from AFM are three-dimensional surface profiles, offering an advantage over the flat, two-dimensional images from other imaging techniques.
The AFM Probe
The probe is regarded as the heart of any AFM setup and comprises the cantilever and tip assembly. Probes are the most commonly replaced part on this type of microscope because&#160; the constant interaction with the samples wears down the tip. Therefore, the choice of material for the probe depends on the properties of the sample. Silicon probes, used to analyze hard samples, are stiffer and sharper than silicon nitride probes, which are better suited to scan softer samples. These sharp tips are produced using electrochemical etching or carbon nanotubes for higher accuracy analysis.
Imaging Modes of AFM
In AFM, surface topography is studied using the interaction between the probe tip and the sample surface. There are two main imaging modes &#8212; a static mode, also referred to as the contact mode, and a dynamic mode.
In the static or contact mode, the tip of the probe is in continuous contact with the sample surface. As the tip drags over the surface, repulsive forces between the sample and the tip result in the cantilever bending, which is recorded. The entire specimen surface is scanned back and forth in both x- and y-axes, called raster scanning, while the vertical movement of the cantilever records the z-axis, thus generating a 3D image.
In the dynamic mode, the probe oscillates just above the sample surface, coming close to, but not touching the surface. Attractive and repulsive forces determine the variation in distance between the tip and the sample, affecting the amplitude of cantilever oscillation. This feedback is recorded to construct the surface topography of the sample.

原子力显微镜

Atomic force microscopy (AFM) is a type of scanning probe microscopy that can analyze topographic details of various specimens like ceramics, glass, ...

在脑核糖凝视与原子力显微镜

摘要

探索更多视频

此视频中的章节

重组DNA和蛋白质复合物，用原子力显微镜的可视化

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

原子力显微镜对单蛋白质分子的力谱

使用原子力显微镜测量活细胞的力学性能

亚纳米级分辨率成像与调幅原子力显微镜在液体

原子力显微镜成像在核细胞结构和动力学研究中的应用

原子力显微镜成像和支持的脂质双层力谱

数百个 C.白化病细胞生物原子力显微镜测量自动化

静态原子力显微镜，用于可视化和表征组装的核小体

原子力显微镜

在脑核糖凝视与原子力显微镜

摘要

探索更多视频

此视频中的章节

重组DNA和蛋白质复合物，用原子力显微镜的可视化

冲红灯光感受器原子力显微镜使用PeakForce定量纳米机械性能映射

原子力显微镜对单蛋白质分子的力谱

使用原子力显微镜测量活细胞的力学性能

亚纳米级分辨率成像与调幅原子力显微镜在液体

原子力显微镜成像在核细胞结构和动力学研究中的应用

原子力显微镜成像和支持的脂质双层力谱

数百个 C.白化病 细胞生物原子力显微镜测量自动化

静态原子力显微镜，用于可视化和表征组装的核小体

原子力显微镜

数百个 C.白化病细胞生物原子力显微镜测量自动化