这项工作是支持部分由美国国立卫生赠款R01 AI067548，R01 GM081520，R01 GM086200，R01 AI049194，和P01 GM066521 GJJ以及霍华德休斯医学研究所，在加州理工学院贝克曼研究所，并从礼品到加州理工学院戈登和贝蒂摩尔基金会和Agouron研究所。 MSL支持由美国国立卫生研究院授予2R37 - A1041239 - 06A1东区S.比约克曼。莱卡微系统公司慷慨地提供视频内容cryosection收集。梅毒螺旋primitia代表结果由Gavin大肠杆菌墨菲的收集和处理，并刊登标题为“梅毒螺旋primitia小说和活力的影响超微结构”分子微生物学。 （墨菲，G.等，为微生物67，1184年至1195年，2008年）。

一，细菌细胞培养制备<ol><li>在其正常的媒体生长的细菌细胞在几毫升液体培养所需的阶段。如果需要浓度较高，纺纱，并可以通过新媒体的再悬浮，但要知道，这个过程有时会介绍了细胞结构的变化。 </li><li>在光镜下检查，以确保文化是无污染物，并在适当的合流。 </li><li>有些细菌的细胞能够坚持，甚至上生长的EM电网。当电网，电网与碳大衣金生长的细胞，是最常用，以避免细胞毒性。他们应该焕发出院2分钟，下一个15分钟的紫外灯灭菌，直接放入液体培养基。贴壁细胞，自然会坚持到网格，和其他人可以提示坚持涂层的电网电网聚- L -赖氨酸0.1％第一。在光镜下检查电网首先，以确保细胞的浓度是适当的冻结前。 </li></ol>二。切入冻结瘦细胞对于薄的细菌细胞，完整的细胞悬浮液是跨EM电网冻结的暴跌。该过程会保留超结构的细菌细胞，并防止水分蒸发后在高真空的EM。这是可以做到定制暴跌冷冻设备，或我们在这里展示与商业冷冻室自动暴跌，飞Vitrobot MKIII 。 <ol><li>辉光放电的碳包覆EM网格2 - 4分钟。这使得电网的表面亲水性，从而帮助均匀地分散到整个电网的样本。 </li><li>混合5％集中BSA溶液25μl100μL胶体金溶液（颗粒直径为10 nm）。 BSA的大衣的金颗粒，并防止其聚集。 </li><li>离心机在18000克BSA和15分钟的黄金粒子混合物。取出上清液后，剩余的黄金颗粒混合与20μL的细胞解决方案。 BSA处理的黄金粒子不与细胞发生反应，但他们将作为基准标记重建的tomograms需要。 </li><li>应用4μL的细胞和金溶液混合EM网格在100％湿度的Vitrobot出院辉光。网格，然后自动抹杀双方NR 1滤纸。这将删除多余的液体，仍然是这样，只有在他们的媒体细胞薄膜。网格，然后迅速下跌到约77K液氮降温的乙烷和丙烷液体混合物。在这个过程中，样品被冻结如此迅速，防止冰晶的形成是在薄膜&lt;1微米。 </li><li>小心取出乙烷/丙烷混合物的电网，并迅速转移到网格储物盒，是根据液氮保存。 </li></ol>三。高压冷冻和低温较厚的细胞切片对于较厚的细胞，高压冷冻减少冰结晶和随后的冷冻切片呈现样本足够薄电磁成像的5，6，7有许多不同的标本持有人可用于高压冷冻，您的选择将取决于样品的类型用于冷冻机或应用程序中选择的调查。在这里，我们使用BAL - Tec的HPM 010高压力冰箱和冷冻超切片模型UC6/FC6徕卡显微系统。 <ol><li>对于细菌，15mls微调文化外径&gt; 0.5采取直接从37℃培养箱中培养，在1000RPM离心3分钟。然后，取出后用20％右旋糖酐冷冻保护剂0.5毫升上清液，余下的文化沉淀的混合0.5毫升在13000rpm离心15秒。 </li><li>去除上清液后，超级颗粒吸管0.1毫升粘贴到热封微量提示已经包含0.2毫升20％右旋糖酐cryoprotecant（40000Mwt）。 13000 RPM为30秒的微管尖端的混合物离心，去除上清液。 5-10μL紧颗粒被认为是在密封的提示。 </li><li>一个“特富龙”涂层的黄铜圆顶planchet在高压冷冻持有人的手臂准备安装接收粘贴在一个半圆顶和与平面伙伴黄铜帽子密封。 </li><li> ARM汇编，然后迅速插入的催芽高压冷冻冻结。 </li><li>含有细胞结合黄铜planchet单位在2100巴的压力正在迅速喷射高压液氮冷却，ARM汇编即刻消除，并陷入液氮浴，防止气候变暖的planchet。 </li><li> Planchet单位储存在液氮，直至冷冻切片。 </li><li>为了揭露对切片的细胞​​冷冻球，两个黄铜planchets仔细分离undeř液氮。 </li><li>细胞完全冻结的圆顶是玻在外观上的裂缝和冰核裂隙。 </li><li>这planchet是转移到预冷的-170 ° C室，并牢固地安装在冷冻切片机夹头副。 </li><li>冷却cryomicrotome包含必要的必要的cryotools，对切片的，其中包括：低角度低温金刚石刀，金刚石修整工具，按平台，网格储物盒，防静电喷涂机和电网控股仪器。 </li><li>对于切片外的圆顶地区的成功色带初步形&lt;0.2毫米平方米的金刚石修整工具和快速的切割速度，同时保留了〜200μm的深度以及冻结周边。 </li><li>在低角度刀和铜在靠近支持电网的防静电喷雾，钻石刀是准备为切片抛光块面仔细先进。 </li><li>切片机的参数选择，如一个切片厚度设置50至350纳米之间，连同〜0.4mm/sec和切割窗口窄的切割速度。重要的是要控制的防静电喷雾的范围和强度低湿度条件下，在不久的面积。 </li><li>由于第一部分是制作了精美的睫毛撒施是操纵手或显微嵌入滑出低角度的钻石刀刃的早期部分。 </li><li>节剪彩，虽然被迫关闭的刀，他们轻轻地支持由睫毛探头，通过附近的铜网支持指导。 </li><li>要坚持的部分，装入用丝带网格，然后坚定地按下使用水冷抛光银探头，部分路段是如此高电荷最新的防静电设备，可协助在粘附过程。 </li><li>电网盒都保存在液氮冷却的干托运人长期贮存，直到在显微镜是电网传输。 </li></ol>四。使用低温光学显微镜检查细胞可以使用的低温光镜下（cryoLM）成像对电网的细菌细胞。我们这里使用的显微镜​​是一个自定义的尼康倒置显微镜TI E / B的超长工作距离（ELWD）60X空气物镜。成像，这是一个改装费冷冻光镜下阶段期间，电网保持在低温阶段。使用取景网格，可位于细胞上的方格和cryoEM tomograms相关cryoLM图像。 <ol><li>装入冷冻站的墨盒电网。墨盒是从我们标准的EM墨盒费TF30H polara修改定制。电网是铜剪辑环。碳粉盒，然后在管在液氮转移中保存。 </li><li>酷液氮温度（-190℃）的低温阶段。 </li><li>墨盒装入冷冻阶段，并移动到网格的观景窗。舞台可容纳多达两个自定义的修改墨盒。 </li><li>下聚光镜和60X物镜聚焦到电网。 </li><li>查找细胞和拍摄图像。对于相关的LM和EM研究，电网扫描周围区域的细胞，定位的细胞后在EM。 </li><li>成像后的网格，把墨盒放回管，并保存在液氮管，直到准备在EM成像。 </li></ol>五，收集倾斜系列在低温透射电子显微镜为了获得一个细菌细胞的三维断层扫描，采取一系列投影图像，而样本是增量沿着一个或两个轴在低温透射电子显微镜倾斜。由于倾斜角度和其他实验设置，有几个不同的软件可以自动收集倾斜系列。我们用我们的低温透射电子显微镜，这是费TF30H polara Leginon。它允许自动倾斜系列预选细胞。 <ol><li>预冷冷冻站的墨盒装入持有多选（MSH）。 MSH最多可容纳6个墨盒。 </li><li> TF30H - polara的MSH的连接，挑一个墨盒，并把它​​插入到EM列。 </li><li>开始对EM计算机和Leginon上的其他工作站的主要方案的Leginon客户端。 </li><li>设置预置（图像条件），为一个Leginon会话。最重要的参数是放大倍率，根据重点的价值，电子剂量和倾斜一系列的倾斜角度，包括起始角度，终止角度和增量。 </li><li>从最低放大倍率开始图像选择的目标，并发送具有较高的放大倍率，他们下一步。 </li><li>排队倾斜系列收集的所有目标，并提交所有这些最后的“断层”的一步。 </li><li>通过从本地工作站的网络工具，可以监视数据收集的过程。 </li><li>实验数据收集完成后，同比下降加载到本地工作站为重建完成的倾斜系列。 </li></ol>六。生物安全考虑大多数的生物样品，我们的工作与非致病性，并已经从土壤，自来水或昆虫的肠道中分离。基本无菌技术操作是足够的处理这些样本。病原体的工作需要额外的安全步骤的实施，但是，对于极端的危害，一些实验室已经安装了整个冷冻电镜在B​​SL - 3实验室显微镜。以下是一些我们减少风险的工作在我们的实验室病原体的的方法。 <ol><li>化学或基因样品的致病性，可以减毒。许多实验室的病毒，做冷冻电镜暴跌冻结之前与化学固定剂灭活。作为替代方案，关键突变可以推出，导致非感染性的样品，包括点突变在某些酶的失活或淘汰受体的实例。 </li><li>应戴手套，实验室外套和护目镜等个人防护装备。 </li><li>有害样品可以在生物安全柜（BSC）的处理。可以生长的细菌培养，浓缩，甚至到新兴市场大跌冻结在BSC电网。我们已经较小，人工插的冷冻设备，到我们BSC的适合于这一目的。如果自动暴跌冰柜提供更大的一致性是必要的，它们可以被引入平衡计分卡，或更简单，在某些情况下，我们支持我们Vitrobot的印迹垫用铝箔，以防止污染设备。与BSC的工作时，许多可能的工具和材料进入或内阁应消毒，用70％乙醇。当我们准备我们的电网，使用小体积的细菌或病毒培养（通常为4 UL），其中大部分得到的Whatman纸吸干。吸墨纸，枪头，和其余的文化作为生物危险废物处置。所有的工具和暴露的表面，用稀释的漂白剂，95％的乙醇，然后水消毒。 </li><li>电网一旦被冻结，他们在整个存储和数据收集液氮温度下保存。虽然，它已被证明，细胞可以生存冻结，然后解冻，所以我们继续来对待他们作为整个存储和数据收集的危险。数据收集，含冷冻电网的墨盒从低温透射电子显微镜和直接下降到70％的酒精。虽然罕见，但应注意到，有时电网“下降”列内，此类事件的后果应为每个危险的样本设想。大多数显微镜在室温，所以在下降电网的样品可能会列内解冻，成为完全的EM超高真空干燥。这显然​​灭活一些生物样品，但也许没有强大的病毒，和干燥样品的残余可能被显微镜列下次打开服务时的气溶胶释放。 </li></ol>七。断层扫描重建细菌细胞tomograms，是通过软件重建。有几个软件，用于这项工作。我们使用自动重建和筛选tomograms的“ 猛禽 ”，9自动倾斜的一系列调整和随后的重建是一个不平凡的任务，猛禽目前不会有100％的成功率。在猛禽出现故障或需要高品质的重建的情况下，我们使用手动图像对齐和与eTomo重建中的IMOD断层软件套件。 ，10，11 <ol><li>从本地Leginon Web服务器的每个下载的个人倾斜系列。保存在一个地方，他们可以很容易地所在的目录。 </li><li>手动检查使用的IMOD的3dmod观者倾斜系列，抛弃那些Leginon已经失败时，跟踪目标，或产生一个有用的倾斜系列。 </li><li>我们运行猛禽分布在实验室中通过桃 （珍珠为基础的系统，通过网络分发任务）在Linux机器。我们指定我们的基准标记的大小，即胶体金颗粒，标记为重建，像素大小，重建所需的厚度，分级的因素和一些基本设置，如输入（输出路径和输出格式在命令行）和离开它运行，一般20分钟至2H 122图像数据集（2K由2K）。如果与重建的结果感到满意，在本节下面的步骤可以跳过。 </li><li>当需要手动重建，重命名“。MRC”文件“。ST”和个人倾斜加载到eTomo GUI在IMOD断层套房系列。 </li><li>指定倾斜系列轴型（单或双轴），并进入SA使用的基准尺寸mple准备。扫描的文件头，以确定像素大小，然后继续点击“创建COM脚本”。 </li><li>在eTomo GUI中的每个选项卡的步骤重建倾斜一系列的断层扫描。检查和核实后，每个步骤的结果，如果不满意，我们可以随时回去，从有到以前的任何步骤，并重新处理。 “前处理”，是执行准备处理和删除异常像素高强度X射线引起的。 “粗对准”产生一个倾斜的系列，其中随后每个倾斜系列投影图像对齐到以前的交叉相关。我们定义的黄金珠使用“基准模型生成”的基准点，并计划在每个投影图像跟踪的基准指标，并建立了“美术对齐”。 “断层扫描定位”允许种植的断层扫描在Z“最终不结盟栈”是使用线性插值和功能，如周大福改正，黄金橡皮擦及2D滤波可以选择性地应用于产生。断层扫描是在“生成断层扫描”Fourier空间中的加权反投影计算。 “后处理”，使种植的断层扫描XY区域的利益和缩放在利益结构的范围对比，并“清理”删除中间文件并释放磁盘空间。 </li></ol>八。存储在数据库中的Tomograms 我们使用一个基于Web的内部数据库来组织，存储，搜索，并分发层析成像数据。对于每个系列倾斜，我们可以记录样品的详细信息，显微镜收集参数，原材料倾斜系列以及相关的三维重建和其他处理的文件。主浏览页面显示缩略图图像，并可以通过下载的频率，创作者，标本类型或日期举办一个有特色的“YouTube”电影就像每个断层扫描和条目。通过输入关键字或特殊功能，用户可以搜索的数据库。目前已超过4500个断层倾斜系列存放在数据库中，占地近60种/标本的类型，共有超过11,000名关联的文件。 九。分析和分割的Tomograms <ol><li> tomograms的结构细节，可以查看和分析，通过不同图像处理工具，如观众与ZAP，XYZ和切片窗口的IMOD 3dmod软件。 </li><li>下一步是创建一个分割的三维图像（断层扫描）。分段分配给每个体素（体积像素）的三维图像的标签描述体素是属于哪个地区或材料，例如，膜或骨架。如IMOD和阿米拉（Visage的影像）3dmod我们使用的软件工具。 </li><li>虽然一些软件工具，提供自动分割模块（例如，阈值分割），他们往往只工作与高对比度的图像。从低剂量电子cryotomography所得的三维图像，低对比度，在大多数情况下，我们必须手动分配一个标签，每个体素。 </li><li>分割存储在一个单独的数据文件。它可用于生成一个表面模型，在每个区域用不同的颜色显示在3D查看。 </li><li> tomograms可用于其他分析。例如，模板匹配和随后的计算，可以产生如核糖体等大分子复杂的统计信息。子卷对齐和平均显示了更高的对比度的结构，并揭示了更精细的细节。 </li></ol>十，基于tomograms电影我们拍电影，总结每一个项目，并给予我们的断层图像的视觉之旅。 <ol><li>做一个脚本在数据的功能，我们想表明，如何使这些功能。例如，在整个细胞的断层项目的一个典型的电影，我们开始通过展示有代表性的倾斜的一系列数据，然后按照一个重建的断层扫描片由片。下一步，我们将展示一个关键大分子在细胞内，例如分割，鞭毛马达或化学感受器复杂或胞质长丝。最后，我们的结论显示一个理想化的解释我们正在研究的大分子模型。 </li><li>阿米拉或嵌合体图形，如代表性的软件是用来呈现电影的个人静帧。我们通常组装较短的剪辑的电影，以方便每个剪辑的变化。 </li><li>使用商业电影编辑软件如Adobe Premiere组装成最终的影片到影片剪辑中的静帧，然后在影片剪辑。 </li><li>我们已经开始添加音轨，叙述了电影。旁白作为“电影传奇”，让观众把重点放在数据一边看电影。 </li></ol>十一。动画视觉讲故事的通信，而无需复杂的词汇具体的科学领域的知识。难以理解的概念变得简单时，伴随着视觉示范。实践说明生物与动画片的想法是不是新的。然而，仅在过去十年中被带到承担建设科学动画的任务的专业3D内容创建复杂的工具。现在有几十个研究人员，积极转化为生物系统的动态三维动画。许多美丽的动画展示<a href="http://MolecularMovies.org">http://MolecularMovies.org</a> 。该网站还举办了教程和分配操纵Autodesk Maya中的分子结构的一个免费的工具包。开源的3D内容套房，搅拌机，包含几个用户生成的PDB进口商。 三维动画的过程一般包括三个步骤： <ol><li>建模：我们使用图形代表性的软件，可以进口到玛雅的格式导出到表面。这些表面重建tomograms之一分割，例如，膜，颗粒剂，或细丝，或分子表面的交涉。在阿米拉断层扫描分割;分子表面生成嵌合体或VMD。原子分子表示可以直接导入到PDB文件玛雅。一旦在Maya的模型，他们可能会被修改，复制，和适当的定位。 </li><li>动画：动画传统手工建造与艺术家手工搬迁移动的物体，并录制了一系列关键帧。三维图形软件中的新进展，现在允许程序动态生成，通过内置的模块和脚本API。 </li><li>渲染：程序动态动态的生物过程，如结构的自组装，可以提供有说服力的动画。然而由于现实地模拟生物系统的困难，三维动画，大部分仍采用传统的关键帧技术。一旦动力，为了视觉的注释，纹理和照明效果可能会增加。最后，相机的路径选择，并完成动画电影中呈现。 </li></ol>

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	<li>Jensen, G. J., Briegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+electron+cryotomography+is+opening+a+new+window+onto+prokaryotic+ultrastructure.">How electron cryotomography is opening a new window onto prokaryotic ultrastructure.</a> Curr. Opin. Struct. Biol. 17, 260-267 (2007).</li><li>Li, Z., Jensen, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography:+a+new+view+into+microbial+ultrastructure.">Electron cryotomography: a new view into microbial ultrastructure.</a> Curr. Opin. Microbiol. 12, 333-340 (2009).</li><li>Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+of+biological+complexity.">Three-dimensional imaging of biological complexity.</a> J. Struct. Biol. 138, 85-91 (2002).</li><li>Iancu, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryotomography+sample+preparation+using+the+Vitrobot.">Electron cryotomography sample preparation using the Vitrobot.</a> Nat. Protocols. 1, 2813-2819 (2007).</li><li>Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J. Microsc. 203, 285-294 (2001).</li><li>Ladinsky, M. S., Pierson, J., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitreous+cryosectioning+of+cells,+facilitated+by+a+micromanipulator.">Vitreous cryosectioning of cells, facilitated by a micromanipulator.</a> J. Microsc. 224, 129-134 (2006).</li><li>Pierson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+technique+of+vitreous+cryo-sectioning+for+cryo-electron+tomography:+Electrostatic+charging+for+section+attachment+and+implementation+of+an+anti-contamination+glove+box.">Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box.</a> J. Struct. Biol. 169, (2009).</li><li>Suloway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated,+sequential+tilt-series+acquisition+with+Leginon.">Fully automated, sequential tilt-series acquisition with Leginon.</a> J. Struct. Biol. 167, 11-18 (2009).</li><li>Fernando, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markov+random+field+based+automatic+image+alignment+for+electron+tomography.">Markov random field based automatic image alignment for electron tomography.</a> J. Struct. Biol. 161, 260-275 (2008).</li><li>Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+visualization+of+three-dimensional+image+data+using+IMOD.">Computer visualization of three-dimensional image data using IMOD.</a> J. Struct. Biol. 116, 71-76 (1996).</li><li>Mastronarde, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-axis+tomography:+an+approach+with+alignment+methods+that+preserve+resolution.">Dual-axis tomography: an approach with alignment methods that preserve resolution.</a> J. Struct. Biol. 120, 343-352 (1997).</li></ol>

我们出现在这个视频文章怎么做ECT的细菌细胞。空间和时间的限制，我们只能显示一般的协议。更多细节，可以发现在论文，网上或其他的设备和软件开发商的制造商提供的信息，和ECT的研究小组。根据不同的细菌种类的研究，所使用的设备，并在细胞的结构利益，协议和实验参数需要测试和优化。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Poly-L-Lysine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P8920</td>
<td>0.1% solution</td>
</tr>
<tr>
<td>MKIII Vitrobot</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gold colloid solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1527-25ml</td>
<td>10 nm</td>
</tr>
<tr>
<td>Bovine serum Albumin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P2489</td>
<td>10%</td>
</tr>
<tr>
<td>Nr 1 Filter paper</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1001 055</td>
<td>&nbsp;</td>
<tr>
<td>Dextran</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>31389</td>
<td>15-25%, MW 39000</td>
</tr>
<tr>
<td>Brass-domed planchet</td>
<td>&nbsp;</td>
<td>Swiss Precision Company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bal-Tec HPm 010 high pressure freezer</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cryo-ultra microtome UC6/FC6</td>
<td>&nbsp;</td>
<td>Leica Microsystems Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Diamond cryo trim 45</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Static line II ioniser</td>
<td>&nbsp;</td>
<td>Diatome US Inc</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CX100 Dry shipper</td>
<td>&nbsp;</td>
<td>Taylor-Wharton-Cryogenics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MSH loading station</td>
<td>&nbsp;</td>
<td>Gatan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ti E/B inverted microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>Cryo LM stage</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>Custom</td>
</tr>
<tr>
<td>TF30-polara</td>
<td>&nbsp;</td>
<td>FEI</td>
<td>&nbsp;</td>
<td>With UltraCam from Gatan</td>
</tr>
<tr>
<td>Amira</td>
<td>&nbsp;</td>
<td>Visage Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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electron cryotomography of bacterial cells

尽管已经称为细菌细胞的基本新陈代谢，许多基本问题仍然令人惊讶的答复，例如，包括他们是如何产生和保持特定的细胞形态，建立极性，分离它们的基因组，并鸿沟。为了了解这些现象，成像技术，桥梁之间的荧光光学显微镜的分辨率和更高分辨率的方法，如X射线晶体学和核磁共振光谱的差距。 电子cryotomography（ECT）是一项新兴技术，它就是这样，使细胞超微结构在附近的原生状态的可视化，三维（3D）与“大分子”的决议（〜4nm），1， 2 ECT的，细胞成像中的玻璃体，“冻结”在一个低温透射电子显微镜在低温（cryoTEM）水合状态（&lt;-180℃）。细长的细胞（高达〜500纳米厚 3），完整的细胞暴跌冻结范围内跨乙烷或乙烷/丙烷混合物的制冷剂，如EM电网的媒体。较厚的细胞和生物膜也可以在玻璃态成像第一“的高压冻结”，然后，“冷冻切片”他们。一系列的二维投影图像，然后通过样品的收集，因为它是逐步沿着一个或两个轴倾斜。一个三维重建，或“断层扫描”，然后可以从图像中计算。近年来，虽然ECT的需要昂贵的仪器，它已在几个实验室使用，以揭示各种外部附属物的结构，结构的信封不同的细胞，细胞骨架细丝的位置和结构，并地点和大大分子结构集会，如鞭毛马达，内部隔层和化学感受器阵 ​​列1， 2 在这个视频文章中，我们说明如何细胞与ECT的形象，包括样品制备过程，数据收集，断层扫描重建，并通过分割和在某些情况下，光镜相关的结果的解释。

Watch this Scientific Journal Video about Electron Cryotomography of Bacterial Cells at JoVE.com

Electron Cryotomography of Bacterial Cells

我们在这里说明如何使用电子cryotomography（ECT）的研究在附近的原生状态的细菌细胞超微结构，“大分子”（〜4 nm）的决议。

细菌细胞的电子Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

electron-cryotomography-of-bacterial-cells

Research

JoVE Journal

Biology

25.3K 次观看.  California Institute of Technology - Caltech. 我们在这里说明如何使用电子cryotomography（ECT）的研究在附近的原生状态的细菌细胞超微结构，“大分子”（〜4 nm）的决议。

视频: Electron Cryotomography of Bacterial Cells

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

使用微流体的细菌的趋化作用的研究 - 访

科研

生物学

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

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

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

测量细菌细胞的抗弯刚度，使用光学陷阱

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

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

暴跌冻结：在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超结构保存细胞内膜蛋白定位电子显微镜分析的CryoAPEX方法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

用于液相电子显微镜的化学固定哺乳动物细胞的石墨烯外壳

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...