作者要感谢在加州大学伯克利分校诺加利斯实验室的成员，在帮助建立初始协议，并在耶鲁大学在他们的帮助王实验室的成员，以建立完整的协议。我们也承认在冷冻电镜设施，并在耶鲁大学医学院的支持高性能计算中心的工作人员。硬件是史密斯一家得奖。

1。随机锥形倾斜方法的原理<ol><li>的随机锥形倾斜方法的原则要求，采取对同一地区内的电子显微镜对标本的显微。一张图片是采取标本untilted位置（图1A）和其他图片是在50至70度（在我们的例子中，我们使用55度）之间的角度倾斜标本。 （图1B） </li><li>使用计算机，数字化显微镜对提出的方方和相同的粒子图像被选中。 （图1C） </li><li> untilted粒子及其倾斜伙伴的图像在三维坐标，彼此相关，倾斜轴的方向和倾斜角度。 （图1D） </li><li> untilted粒子图像的对齐方式带来的图像倾斜颗粒，其相应的方位角位置。 （图1E） </li><li>使用多个图像倾斜颗粒填充方位的空间，分子的三维结构，可使用背投影算法重建。 （图1F） <img src="/files/ftp_upload/2574/2574fig1.jpg" alt="图1" /> 图1。RCT的重建原则的一个例证。 </li></ol> 2。准备薄碳的多孔碳电网理由：我们使用负染色法的蛋白质样品随机锥形倾斜重建修复。为了保护没有太多干燥压扁的大分子，我们尝试嵌入在厚度蛋白4维深染色的蛋白质分子。一般情况下，连续碳负染标本。然而，这种碳是难以控制的蛋白颗粒周围的色斑厚度。因此，我们使用自制的多孔碳电网覆盖一层薄薄的碳薄膜（〜5 nm厚）负染标本。由孔形成的小水井允许保留的蛋白质溶液和对电网的染色解决方案，所以它是非常容易嵌入到一个最佳的染色厚度的蛋白质。此外，一层薄薄的碳孔，大大降低了背景噪音。 <ol><li>准备0.5％的Formvar溶液。在通风柜中，添加0.45克聚乙烯醇缩甲醛树脂在100毫升的玻璃烧杯和90毫升氯仿。使用铝箔覆盖烧杯中，并用一个小的轰动吧，帮助溶解在磁力搅拌器的正式树脂。它大约需要15分钟，溶解树脂。 </li><li>在解散的Formvar，清洁的显微镜玻片在甲醇与Kimwipes擦拭干。 </li><li>后For​​mvar树脂完全溶解在氯仿中，加入1 mL 50％的甘油溶液的表面。调整添加甘油的体积影响多孔碳的孔密度。在解决方案ultrasonicator的一角浸在大约1英寸的深度和使用的最大功率超声1分钟，使Formvar解决方案中的甘油滴乳液。该解决方案成为这一步后乳白色。较长的超声导致更小尺寸的多孔碳孔。 </li><li>超声后立即蘸清洗载玻片垂直插入乳液为1秒，带他们出去，和幻灯片的底部，在幻灯片表面形成薄薄的塑料薄膜用滤纸吸干。 choloroform蒸发后，光学显微镜下检查在影片中孔的密度和大小。根据需要调整的准备条件。在这里，我们与条件普遍获得孔，直径3〜4米和10〜20孔，每平方米的400网格。 </li><li>玻片干燥后，剪切滑动面的塑料薄膜的边缘。浮膜蒸馏水的表面上。在水面上的薄膜，可以观察到对光线的反射角度一瞧。广场电影逐一与网格“的顺利表面朝下，400目铜网格。 </li><li>拿起一张纸，就可以使用电网的塑料薄膜。翻转纸张，在培养皿让它干。浸泡在甲醇中的文件删除孔中残留的甘油，并让纸张在空气干燥。 </li><li>大衣电网的碳层厚度〜20纳米碳蒸发器。厚度可确定的碳灰色。 </li><li>在氯仿中浸泡半个小时删除的Formvar碳的网格。电网干燥后，我们已取得自制的多孔碳电网。 </li><li>一层薄薄的碳挥发到新鲜剥离的云母表面，厚度约5纳米。 </li><li>小心翼翼地把蒸馏水下面的多孔碳电网。浮法从云母表面的水面上薄薄的碳沉积到多孔碳电网缓慢。在通风橱中干燥的网格。 </li></ol> 3。外切体复合物的负染理由：有不少重金属染色解决方案，可为负染色的EM使用，包括醋酸铀，铀甲酸，磷钨酸，钼酸铵，和其他人。不同的污渍溶液具有不同的独特属性。例如，醋酸铀粒子的高对比度，但可能会崩溃，不喜欢酸性环境中的蛋白质复合物。对于这些样本，在中性pH phosphotungestic酸可能是一个良好的污点解决方案。我们选择饱和铀酸盐（UF）解决方案，由于其粒度小，成分子的高渗透能力。 <ol><li>蒸馏水煮沸1分钟。慢慢冷却下来到室温。这一步是去除水中的溶解氧。 </li><li>让新鲜的2％的铀酸盐（UF）解决方案。在1.5 mL试管中混合1 mL水和20毫克用友。涡为10分钟。 </li><li>加入2微升10米的氢氧化钾调节pH值至5.0。混合立即。溶液的颜色应该是黄色。溶液的pH值不应该太高，否则污渍沉淀。 </li><li>再过10分钟就vortexer管。 </li><li>自旋在台式离心机最大速度为10分钟的解决方案。 </li><li>通过0.2微米的聚偏氟乙烯膜的过滤解决方案。这是新鲜的用友的解决方案。一块铝箔覆盖的解决方案，以防止光管。该解决方案将在同一天使用。 </li><li>辉光放电一薄层碳过多孔碳网格，使用辉光放电装置，在25 mA电流为30秒。 </li><li>放在板凳上了一块干净的封口膜。把3液滴的50微升用友顶部的封口膜染色的解决方案。 </li><li> 50〜100纳米稀释缓冲液（25毫米的Tris - HCl pH值7.5，氯化钠100毫米，2毫米数码地面电视）的浓度稀释的外切体的复杂。把4微升稀释蛋白辉光放电电网。让样品留在一分钟的网格。 （注：这种分子终浓度一般给出了一个最佳的负染电网以及分散的颗粒密度甲酸铀或铀acecate染色解决方案，一般的磷酸盐或高浓度的盐（超过0.5米）没有获得良好的染色效果好，我们的经验表明，HEPES或管道的工作以及与铀染色解决方案。） </li><li>使用滤纸印迹从电网的边缘残余的解决方案，并立即翻转染色液滴上的电网，每个液滴冲洗约10秒钟的电网。 </li><li>最后一次冲洗后，让另1分钟污渍停留，然后对电网的印迹染色滤纸。保持网格表面上的污渍溶液的薄薄的一层，以获得良好的深染色结果。让电网的快速干燥在通风柜。 </li></ol> 4。外切体复合物的电子显微镜理由：任何一个倾斜的阶段透射电子显微镜可用于收集标本RCT的重建倾斜对。在理论上，更高的角度标本可倾斜来收集数据，更好。在实践中，由于试样座和电网的几何设计，最高可操作性的角度是有限的，从50到70度。在这个协议中，我们只说明我们的程序使用一个费Tecnai - 12电子显微镜。对于其他型号的显微镜，操作需要进行调整，根据项目的要求和仪器的财产。 <ol><li>样品架的样品电网，然后放入一个FEI Tecnai -12电子显微镜持有人。显微镜是在120千伏运行。我们使用加坦Ultrascan4000 CCD相机拍照。确保“配置相机的数码显微接口”对话框中的“围绕垂直轴翻转”是未经检查，以确保正确的霸道决心。 （注：这是很重要的，尤其是如果读者依赖蜘蛛RCT的重建过程，得到一个三维模型。） </li><li>检查样品电网在低倍率找到最好的染色广场。这类广场应该有十几孔与维约1〜2微米，并在他们的深色染色领域。 （图2） <img src="/files/ftp_upload/2574/2574fig2.jpg" alt="图2" /> 图2：一个显示好的和坏的污渍的正方形格子的一个低倍率的显微镜。 </li><li>打开了费的用户界面的低剂量模式和调整搜索，重点和暴露在低剂量模式的地位。我们使用的风险，并为搜索衍射相机长度1.5米，52000重点，放大倍率15万。调整曝光时间为1秒。远离暴露POS设置焦点位置2微米ition沿倾斜轴。 </li><li>查找与搜索模式染色的孔，并保存的位置。良好的孔一般有深色斑梯度搜索模式下观察。以一个正方形的CCD图像。试样倾斜至55度，拍摄另一张照片。比较两个图片，找出配对的两个显微孔。 （图3） <img src="/files/ftp_upload/2574/2574fig3.jpg" alt="图3" /> 图3。倾斜对阿方在搜索模式的显微照片。相应的配对孔表示。 </li><li>回到0度倾斜的舞台。使用低剂量的套件，以确定在搜索模式中每孔在高放大倍率的照片。使用的散焦-0.7微米左右。 </li><li>毕竟洞已经采取的图片，倾斜至55度的阶段。采取同一放大倍数为约-1.2微米离焦untilted的倾斜试样的显微照片。确定在低倍率显微模式为基础的倾斜，对相应的显微照片。自制的多孔碳电网的不规则图案的相关帮助。检查倾斜对显微和删除坏污渍，如浅染区（颗粒出现倾斜条件下他们周围的光环）的显微照片。 （图4） <img src="/files/ftp_upload/2574/2574fig4.jpg" alt="图4" /> 图4。倾斜对标本在高倍显微。好的和坏的显微照片标记。 </li></ol> 5。图像处理的数据理由：有不同的选项和软件包，执行电脑中的随机对照试验的重建。最普遍使用的是5天基。一个基本的协议来执行RCT的蜘蛛，在<a href="http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html">网页</a> http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html中可以找到。一个详细的协议来执行RCT的蜘蛛， 谢赫等人在文章中描述 ，在我们的协议 6，我们使用IMAGIC - 5 7，在该协议的视频版本蜘蛛的组合。我们还提供了一个替代的过程，仅仅用蜘蛛在该协议的文本版本。 <ol><li>设定的程序。我们使用电子舱单8封装的proc2d加坦数字图像，改变图像格式蜘蛛的图像格式。 IMAGIC - 5是用来做2D对齐。天基信息平台是用来做三维重建和完善。 </li></ol>第1：采摘倾斜对粒子。 <ol start="2"><li> *. DM3加坦数字图像转换成以天基信息平台的图像格式使用电子舱单proc2d命令。倾斜和untilted对被命名为模式*** t.spi和*** u.spi。 </li><li>挑选颗粒对使用“网”计划，分布在蜘蛛程序包。在按照指令坐标全自动untilted和倾斜颗粒分别保存在DCU ***. SPI和DCT ***. SPI。另一份文件DCB ***. SPI包含倾斜角倾斜和untilted颗粒之间的信息（注：在装修中的三个角度之间的倾斜双网不保证最终重建正确的霸道，因为THETA没有符号（正值）和phi和伽玛依靠设置为它们的初始值，检查的散焦渐变的方向倾斜显微帮助设置正确的披和伽玛装修前的初始值，以得到正确的霸道的重建图5说明正确的公约。） <img src="/files/ftp_upload/2574/2574fig5.jpg" alt="图5" /> 图5：在WEB倾斜对粒子采摘散焦渐变的装修模式决定了一个例子来解释角度决心公约。 H代表高散焦区，而L代表低，散焦区。箭头代表倾斜轴。为每个计划的PHI和γ相应的正确的初始角度表示。 </li><li>开箱使用天基信息平台脚本修改后的版本在网页所示的挑颗粒<a href="http://www.wadsworth.org/spider_doc/spider/docs/partpick.html">http://www.wadsworth.org/spider_doc/spider/docs/partpick.html</a> 。脚本保存u.spi和t.spi untilted和倾斜颗粒堆栈。应保存在particle_list.spi粒子显微图像的数字。 （注：生成的正确的欧拉角文件，这是非常重要的RCT的。） </li></ol>小组第2条：untilted粒子图像的二维排列和分类。 <ol start="5"><li> IMAGIC - 5格式转换成untilted颗粒em2em计划在美锜- 5封装。对齐和反复使用的IMAGIC - 5方案（附录A）分为同质类的粒子。使用IMAGIC - 5 MSA名级的命令来生成类的粒子，我们保存为imagic_classes.lis查找表。 （注：分类和排列形状相同的粒子数增加，以及减少在一个类的变化差异。每个类的地图可以提供一流的质量信息。） </li><li>每个粒子使用IMAGIC - 5头命令对齐的平移和旋转值生成一个图形文件（视频演示ali_50.plt）。 </li><li>转换的类查找到蜘蛛文档文件base_file ***. SPI表使用一个Perl脚本lis2spi.pl在分布式<a href="http://cryoem.berkeley.edu">http://cryoem.berkeley.edu</a> 。 </li><li>转换每个粒子从翻译和旋转值到5.6天基信息平台的文档文件使用脚本plt2spi.pl在分布式ali_50.spi步生成的图形文件对齐的平移<a href="http://cryoem.berkeley.edu">和</a>旋转值http://cryoem.berkeley.edu。 我们使用2D的调整和分类IMAGIC - 5，因为它使这项工作在我们手中的最佳性能。蜘蛛，替代战略的二维排列和分类 ​​，可以发现<a href="http://www.wadsworth.org/spider_doc/spider/docs/align.html">在</a> http://www.wadsworth.org/spider_doc/spider/docs/align.html 。我们也用蜘蛛来执行外切体的untilted颗粒的二维分析。以下是一个简单的程序。 使用替代5.5）中所描述的参考免费对齐对齐图像。两个简单的脚本可以发现保存在一个文档文件angular_file.spi中的所有粒子的旋转和移位。 替代5.6）分类成组对齐颗粒中所描述的相同的看法我们已经使用的K - means分类方法。生成base_file ***. SPI的分类的基础上。 </li></ol>小组第3条：使用倾斜的粒子图像三维重建。 <ol start="9"><li>带通滤波器，面具和中心倾斜颗粒就像untilted IMAGIC - 5颗粒。生成一个新的数据集名为颗粒。 （注：这一步是可选的蜘蛛，它可以做。） </li><li>创建anglular类查表的文件ali_50.spi，DCB ***. SPI步骤5.3和步骤5.4粒子的列表文件par​​tile_list.spi使用在附录B中的“蜘蛛脚本的网页所产生的文件的文档文件</li><li>使用蜘蛛，重建脚本中描述<a href="http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html">的</a> http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html做到每班重建。每类颗粒有助于一个三维重建体积。三维模型，可以研究在加州大学旧金山分校的奇美拉9。一个欧拉角（0,0,0）的三维模型的二维投影可以从untilted粒子到相应的2D类平均来验证重建的质量。查找类似卷，调整和合并生成下面的过程初始卷<a href="http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html">http://www.wadsworth.org/spider_doc/spider/docs/techs/rancon/recn.html</a> 。 </li></ol>小组第4部分：使用untilted粒子图像三维重建的细化。 <ol start="12"><li>做投影的合并对所有的untilted粒子的初始音量匹配细化，以获得更高的分辨率volumE没有丢失锥和使用天基信息平台的脚本中所述扁平神器<a href="http://www.wadsworth.org/spider_doc/spider/docs/techs/recon/refine.html">http://www.wadsworth.org/spider_doc/spider/docs/techs/recon/refine.html</a> 。 </li></ol> 6。代表性的成果： 使用RCT的方法，我们已获得约50的外切体的重建，从5000倾斜对总（图6）。从50三维模型，我们可以看到主要有两个正交视图对电网的复杂坐在不同的取向。一个扁平化的神器也检测到在许多碳表面垂直的方向卷。我们执行的路线和合并的3D卷生成两个正交视图的初始卷。使用的5000 untilted粒子图像，我们已获得约18埃的分辨率相同的外切体的三维重建，最终来自初始模型（图7）。该结构揭示酵母外切体的架构，并提供 10到RNA底物的招聘途径的见解。 <img src="/files/ftp_upload/2574/2574fig6.jpg" alt="图6" /> 图6 50 RCT的重建外切体的复杂的三维模型。 <img src="/files/ftp_upload/2574/2574fig7.jpg" alt="图7" /> 图7。细化后的外切体复合体的三维重建。 附录： 附录A 2D对齐和分类IMAGIC - 5的脚本文件。 文件：auto_align_i.sh <a href="http://www.jove.com/files/ftp_upload/2574/auto_align_i.sh">点击这里的文件</a> 附录B生成三维重建，蜘蛛，角文件的脚本文件。 文件：generate_angular_file.spi <a href="http://www.jove.com/files/ftp_upload/2574/generate_angular_file.spi">点击这里的文件</a>

<ol>
	<li>van Heel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angular+reconstitution:+a+posteriori+assignment+of+projection+directions+for+3D+reconstruction.">Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction.</a> Ultramicroscopy. 21, 111-123 (1987).</li><li>Radermacher, M. <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+reconstruction+of+single+particles+from+random+and+nonrandom+tilt+series.">Three-dimensional reconstruction of single particles from random and nonrandom tilt series.</a> J Electron Microsc Tech. 9, 359-394 (1988).</li><li>Penczek, P. A., Grassucci, R. A., 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=The+ribosome+at+improved+resolution:+new+techniques+for+merging+and+orientation+refinement+in+3D+cryo-electron+microscopy+of+biological+particles.">The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles.</a> Ultramicroscopy. 53, 251-270 (1994).</li><li>Ohi, M., Li, Y., Cheng, Y., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+staining+and+image+classification+-+powerful+tools+in+modern+electron+microscopy.">Negative staining and image classification - powerful tools in modern electron microscopy.</a> Biol Proced Online. 6, 23-34 (2004).</li><li>Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., Leith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDER+and+WEB:+processing+and+visualization+of+images+in+3D+electron+microscopy+and+related+fields.">SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields.</a> J Struct Biol. 116, 190-199 (1996).</li><li>Shaikh, T. R., Gao, H., Baxter, W. T., Asturias, F. J., Boisset, N., Leith, A., 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=SPIDER+image+processing+for+single+particle+reconstruction+of+biological+macromolecules+from+electron+micrographs.">SPIDER image processing for single particle reconstruction of biological macromolecules from electron micrographs.</a> Nat Protoc. 3, 1941-1974 (2008).</li><li>Heel, M. v. a. n., Harauz, G., Orlova, E. V., Schmidt, R., Schatz, M. <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+generation+of+the+IMAGIC+image+processing+system.">A new generation of the IMAGIC image processing system.</a> J Struct Biol. 116, 17-24 (1996).</li><li>Ludtke, S. J., Baldwin, P. R., Chiu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMAN:+semiautomated+software+for+high-resolution+single-particle+reconstructions.">EMAN: semiautomated software for high-resolution single-particle reconstructions.</a> J Struct Biol. 128, 82-97 (1999).</li><li>Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., Ferrin, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCSF+Chimera--a+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera--a visualization system for exploratory research and analysis.</a> J Comput Chem. 25, 1605-1612 (2004).</li><li>Wang, H. W., Wang, J., Ding, F., Callahan, K., Bratkowski, M. A., Buttler, J. S., Nogales, E., Ke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+yeast+Rrp44+exosome+complex+suggests+routes+of+RNA+recruitment+for+3'+end+processing.">Architecture of the yeast Rrp44 exosome complex suggests routes of RNA recruitment for 3' end processing.</a> Proc Natl Acad Sci USA. 104, 16844-16849 (2007).</li><li>Scheres, S. H., Nunez-Ramirez, R., Sorzano, C. O., Carazo, J. M., Marabini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+for+electron+microscopy+single-particle+analysis+using+Xmipp.">Image processing for electron microscopy single-particle analysis using Xmipp.</a> Nat Protoc. 3, 977-990 (2008).</li><li>Yoshioka, C., Pulokas, J., Fellmann, D., Potter, C. S., Milligan, R. 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在这篇文章中，我们提出了一个详细的协议的样品制备和3负染色电子显微镜的外切体的使用复杂的三维重建。使用这种方法，我们获得了使用随机锥形倾斜的方法没有任何先验知识结构的三维重建。随机锥形倾斜的方法并不一定要求均匀的样品，但下面的投影匹配的细化步骤需要均匀的试样，以实现高分辨率。 负染样品，污渍的厚度是非常重要的随机的锥形倾斜的方法来工?...

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<td>Polyvinyl Formal Resin</td>
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single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method

单粒子电子显微镜（EM）重建最近已成为一种流行的工具来获得大量的大分子复合物的三维（3D）结构。 X射线晶体相比，它具有一些独特的优势。首先，单粒子电磁重建不结晶的蛋白质样品，这是在X射线晶体学的瓶颈，尤其是对大量的大分子复合物。其次，它并不需要大量的蛋白质样品。单粒子电磁重建毫克蛋白质结晶必要相比，只需要在纳米摩尔浓度的蛋白质溶液的几个微升，使用的EM方法的负染色。然而，尽管少数具有高对称性的大分子集，单粒子EM是有限的分辨率相对较低（低于1纳米的分辨率），尤其是那些没有对称的许多标本。这种技术也由所研究的分子大小的限制，即100 kDa的负染标本和300 kDa的一般冷冻水合标本。 对于一个未知结构的新样品，我们一般使用重金属解决方案嵌入负染色分子。然后，该标本是在透射电子显微镜检查，采取二维（2D）分子的显微照片。理想的情况下，蛋白质分子有一个同质化的三维结构，但表现出不同方向的显微照片。这些显微照片的数字化和计算机处理“单粒子”。用二维的调整和分类技术，在相同的看法同质分子聚集成类。其平均值提高的信号分子的2D形状。我们分配适当的相对取向（欧拉角）的颗粒后，我们将能够2D粒子图像重建成三维虚拟卷。 在单个粒子的三维重建，一个必不可少的步骤是正确分配每个单粒子的正确方向。有几种方法来分配每个粒子的看法， 包括 1角的重整和随机的锥形倾斜（RCT）方法2。在这个协议中，我们描述了在我们的实践，得到酵母外切体采用负染色EM和Rct复杂的三维重建。应该指出的是，我们的电子显微镜和图像处理的协议如下RCT基本原则，但不是唯一的方法来执行的方法。我们首先介绍如何嵌入到一个铀酰甲酸层的厚度相媲美的蛋白质大小的蛋白质样品，使用多孔碳电网覆盖着一层连续的薄碳膜。 untilted（0度）和倾斜（55度），将用于处理和获取初始酵母外切体的三维模型后的显微对，然后插入透射电子显微镜，收集标本。为此，我们进行RCT和再细化匹配细化方法3使用投影初始的3D模型。

Watch this Scientific Journal Video about Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method at JoVE.com

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

本文介绍了一种标准的方法来获得一个生物大分子的三维（3D）重建使用负染色电子显微镜（EM）的。在这个协议中，我们将解释如何使用随机的锥形倾斜重建方法（RCT）的决议中等酿酒酵母外切体复杂的三维结构。

单粒子电子显微镜外切体综合重建，使用随机的锥形倾斜方法

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

single-particle-electron-microscopy-reconstruction-exosome-complex

Research

JoVE Journal

Biology

23.4K 次观看.  Yale University. 本文介绍了一种标准的方法来获得一个生物大分子的三维（3D）重建使用负染色电子显微镜（EM）的。在这个协议中，我们将解释如何使用随机的锥形倾斜重建方法（RCT）的决议中等酿酒酵母外切体复杂的三维结构。

视频: Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

科研

生物学

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

衣藻中心粒单粒子重建的分离与荧光成像

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

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 ...

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals.

通过共聚焦显微镜重建单细胞先天荧光特征

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

阵列断层扫描工作流程，用于使用扫描电子显微镜有针对性地采集体积信息

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

使用新型机器人系统 Spotiton 进行时间解决研究的低温电子微观网格准备

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Direct Stochastic Optical Reconstruction Microscopy of Extracellular Vesicles in Three Dimensions

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often require indirect methods due to their small diameter (40-250 nm), which is beneath the diffraction limit of typical light microscopy. We have developed a super-resolution microscopy-based visualization of EVs to bypass the diffraction limit in both two and three dimensions. Using this approach, we can resolve the three-dimensional shape of EVs to within +/- 20 nm resolution on the XY-axis and +/- 50 nm resolution along the Z-axis. In conclusion, we propose that super-resolution microscopy be considered as a characterization method of EVs, including exosomes, as well as enveloped viruses.

Direct stochastic optical reconstruction microscopy (dSTORM) is used to bypass the typical diffraction limit of light microscopy and to view exosomes at the nanometer scale. It can be employed in both two and three dimensions to characterize exosomes.

三维细胞外囊泡的直接随机光学重建显微镜

Extracellular vesicles (EVs) are released by all cell types and play an important role in cell signaling and homeostasis. The visualization of EVs often ...