We thank the kind donations of AAV5 sample by Mavis Agbandje-McKenna and Antonette Bennett, and of TMV sample by Ruben Diaz. This work is supported by American Heart Association grant 14GRNT19660003 and VCU startup funds to MS.

1.低温持有人编写和维护注:干抽站，包括一个涡轮增压泵站和一个或多个耐寒级控制器，主要用于三个目的:1）它是一个安全的地方存放冷冻持有人时，不使用它们。正确的方式来存储持有人是在真空条件下让他们在车站。 b）向蒸发时，液体氮从杜瓦和低温保持器的尖端露出去除时发生的霜冻和湿度堆积;这是与热身周期来实现的。 c）向再生沸石干燥剂，并实现了高真空中cryoholder杜瓦瓶。这是必要的操作的低温电子显微镜时保持一个恒定的温度。 <ol><li>预热循环。 <ol><li>将冷冻架成的泵站抽水的港口之一。挂钩疏散管子保持器的真空阀下方的金属出口中的一个。使确保在站（V1，V2和V3），并在低温保持器的所有阀都关闭。 </li><li>转冷阶段控制器上，它通过控制线连接到所述支架。转动涡轮泵站的电源开关。 </li><li>开始在寒冷的阶段控制器的热身周期选项。当涡轮泵站真空读取10 -3托或更好，打开蝶阀V2，等到真空稳定，然后打开蝶阀V1。 </li><li>运行预热周期为约30分钟，直至在保持器温度稳定在20℃以上。关闭V1，然后停止循环。 </li></ol></li><li>沸石周期。运行在4小时和澳沸石周期/ N前CryoEM会议，并每周一次，如果不使用。 <ol><li>启动沸石周期选项，并选择必要的时间，以达到良好的真空，在1-2×10 -4乇的范围内，从而保持时间长久。当真空度达到10 -3托，打开杜瓦疏散阀，V3。那么泵站将撤离线持有人;等到真空稳定。 </li><li>打开在低温持有者阀门。当周期完成后，关闭阀门在它们被打开了相反的顺序:阀门的持有人，那么V3，V2，终于V1上。关掉涡轮泵站和冷阶段控制器，并且从冷阶段控制器和涡轮泵站断开低温保持器。 </li></ol></li></ol> 2.电网准备<ol><li>清洁。 注意:当使用商业多孔网格，它是建议清除它们在使用前，为了除去被在制造过程中使用的任何残余的聚合物。在玻璃培养皿5层滤纸铺设在底部做到这一点。 <ol><li>放置网格上与所述多孔碳的一面朝上的滤纸。 </li><li>使用玻璃巴斯德吸管，湿纸用几滴Ø各地电网˚F氯仿，直到他们刚开始浮动。 </li><li>覆盖培养皿的盖子在通风橱O / N微微张开。 </li></ol></li><li>注:所述薄的碳载体（步骤2.2-2.3）可以随着冰层能直接穿过小孔的多孔碳形成的网格被省略。 <ol><li>用一对镊子，切割一块云母揭露了两个新的表面。放置的云母面朝上新暴露的表面上的白纸在培养皿。 </li><li>放置在培养皿中钟罩碳蒸发器。启动抽空序列，直到真空度低于2×10 -6托。以蒸发碳棒的表面上的任何杂质，进行预蒸发的培养皿盖。然后在空气中的钟罩，并从培养皿取下盖子。 </li><li>直到真空度低于10 -6乇和外套带有一个薄的碳层（〜5nm）的云母进行抽空序列。碳过程中使用保护眼睛蒸发。通过在纸张上产生的黑暗已被放置在云母片下监视所述碳膜的厚度。 </li></ol></li><li>转移薄的碳的TEM网格。 <ol><li>切一个0.5×1cm的片涂覆的云母和浮碳关上的过滤，去离子水的平坦面。使用光学镜头纸撒气水面。使用镊子，将网格的多孔碳侧与浮动碳层的上表面接触，并把它捡起来。 </li><li>直到网格足够数量的准备重复步骤2.3.1和2.3.2。 </li></ol></li><li>辉光放电。 注:辉光放电的网格的天然疏水性碳涂层转化为亲水性的。此步骤是可选的。 <ol><li>将与碳面朝上铺着封口膜7.5×2.5厘米的玻璃幻灯片上的网格。将其放入辉光放电设备的室内，关闭室。 </li><li>泵钟罩和辉光放电20秒，在25 mA和7×10 -2毫巴。 注意:在此期间，一个浅紫色辉光变得可见。与网格取出载玻片并在1小时内使用它们，否则，它们将需要再次辉光放电。 </li></ol></li></ol> 3.样品切入冻注:使用本仪器，以防止液态氮和乙烷喷溅时，请使用防护眼镜和适当的鞋类。 <ol><li>关闭暴跌冷冻设备上。填写加湿器水库用蒸馏水。设定所需温度，相对湿度和印迹时间切入条件，吸干力和吸附时间（22℃，95％，2秒，2和40秒在这里介绍的实施例）。将新的印迹滤纸。 </li><li>通过用液氮填满外侧贮稳定性直到达到（约10分钟）冷却乙烷容器向下。一旦液氮停止boilin克，放置管从乙烷罐中的内室中来的前端和打开阀门非常缓慢。液化乙烷的内室中，并将其填充至1mm从顶部。避免冻结乙烷。注意:乙烷是非常易燃，易爆。 </li><li>确保镊子的暴跌冷冻设备中对齐。 </li><li>打开湿度。加载网格上镊子，并加载3-5微升样品到多孔格栅的碳表面（暗面）。等待将样品吸附到网格并执行印迹来实现的薄液体层。 </li><li>通过浸入入液态乙烷闪冻电网。从液体乙烷保持镊子稳步取出镊子栅组件，并将其与液氮传输到外室。网格转移到一个小格子框浸没在液氮中。确保传递给任一贮槽期间保持玻璃化样品在液氮中的所有时间或往低温保持器。 注:网格拖曳可以存储在一个大容量（35-50升）液氮杜瓦至少1年。存放方便可以将它们放置在50毫升的Falcon管中有两个钻孔的顶部和附加到其上限可以从杜瓦口中被拉出的钓线。 </li></ol> 4.将冷冻格的TEM <ol><li>将冷冻持有者进入低温转移工作站。小心不要插入过程中会损坏夹持器的前端。检查小纤维的存在和灰尘在杆和低温保持器的使用放大镜的O形环，如果存在任何具有光学镜头纸将其删除。 </li><li>填充低温保持器的杜瓦和用液氮工作站容器中。避免使用被困漏斗附带工作站溢出的液氮。 </li><li>在低温持有人连接到寒冷的阶段控制器监视温度，并不断添加液氮＆＃160;待温度稳定在大约-194℃。确保液态氮的水平下都在工作站容器和低温保持器杜瓦真空密封的水平。预冷却该网格镊子，夹子环和工作站上的剪辑环工具的钝端。还预先冷却的一组大镊子和在另一杜瓦充满液氮螺丝刀。 </li><li>迅速使用大镊子在工作站装有冷冻水合电网的冷冻格框转移到液氮中。用螺丝刀打开冷冻格中，采取冷冻水合电网，并把它放入样品架插槽。 </li><li>将在网格顶部的夹子环，轻轻按下它用夹子环工具的钝端，直到其牢固就位就位。关闭冷冻快门。从工作站删除工具和冷冻格箱。 </li><li>移动与固定器和网格显微镜控制台整个工作站，以小迈兹网格在室内空气的传送时间。 </li><li>顶掉的TEM防脏物用液氮，其中已预先填充并冷却至少20分钟。预倾斜显微镜阶段至-60℃。 注意:作为保持器被插入到气密室这将最大限度地减少液态氮的损失。 </li><li>开始在显微镜预泵气闸循环。确保该阀到电子枪被关闭（特别是如果它是一个FEG TEM）。等待预先泵气密室序列插入低温保持器之前完成（约2分钟）。 注意:这由红灯在舞台上被扑灭。 </li><li>将持有人进入气闸舱，并旋转90°顺时针;寒冷的阶段，控制器线连接到冷冻持有者监测温度。再等到红灯熄灭，旋转所处的阶段和持有人回0°位置向持有人插入到TEM的喜GH真空柱。 </li><li>确保温度从不高于-160℃，以避免形成结晶冰。 </li><li>笔芯的低温持有人的杜瓦。等待15 - 45分钟，直至持有人热平衡和TEM真空已录制图像之前恢复。 </li><li>如果检测到液氮鼓泡，改变液体充氮气的高度，或轻轻触摸冒泡的起源，用棉签。 </li></ol> 5.低剂量的数据收集注:低剂量的数据收集技术用于通过限制暴露于20电子/ A 2，以尽量减少电子辐射损坏样品。这是通过三种配置之间交替实现:"搜索"，用低倍率（5000倍〜）和看来，"重点"宽领域，具有较高的放大倍数（〜150,000X）和视野小，而"曝光"，与所需的最终放大倍率fication和含有感兴趣的区域，以进行记录。空白模式之间的光束。 <ol><li>成立了TEM和低剂量计划<ol><li>缩回冷冻快门并打开柱阀。 </li><li>中心舞台，并使用alpha摇摆摇滚舞台±15°设置eucentric高度（Z）。调整的Z位置，直到有而阶段摆动没有明显的移动。激活低剂量程序，并选择检测器的每个模式。 </li><li>在曝光模式找到具有无碳网格的一个区域中，通过选择最佳聚光光圈和斑点尺寸，使得被记录的区域被充分照亮调整照明。一定要传播光束在overcondensation方向。 </li><li>在搜索模式，找到一个小功能，这将是很容易识别，在更高的放大倍率。在曝光模式，围绕这个功能用操纵杆（XY工作台控制器）。开始以较低的放大倍率如果壮举URE不在视场中。 </li><li>在搜索模式中，把该功能来使用低剂量的xy图像偏移控制场的中心。去曝光模式和移动部件沿倾斜轴使用操纵杆，直到它出要被记录的区域（0.5 - 3微米）。转到对焦模式，并使用xy图像移位控制中心的功能。开始在较低的放大倍率，如果该功能不能在视场。 </li><li>保持居中在任何时候都光束。 </li></ol></li><li>数据收集<ol><li>缩回冷冻快门并打开柱阀。激活低剂量程序。 </li><li>在搜索模式，识别含有薄，均匀的冰方格。 </li><li>选择一个合适的孔，并把它在该领域的中心。切换到对焦模式和对焦图像。然后underfocus 0.5之间的图像 - 4.5微米。 </li><li>切换到曝光模式，并记录该图像。 </li><li>切换到搜索模式，并重复步骤5.2.3-5.2.4。移动越过方格避免预曝光好孔以进行记录。 </li><li>完成方格，并移动到另一个不错的。 </li><li>调整高度（Z）和继续数据采集。 </li></ol></li></ol>

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The authors have nothing to disclose.

TEM接着SPA贡献许多大分子的三维结构，即将在EMDB 2000项由2014年中期一般来说，对于给定的大分子，低分辨率3D结构首先从NS数据，其可以随后是higher-确定分辨率cryoEM 3D结构。通过生理盐水，这有利于所述第一三维重建所提供的分子边界的高对比度，随后精制cryoEM，它能够与大分子的内部结构映射在其完全水合状态，并达到更高的分辨率的可能性。 cryoEM的另一个优点是消除脱水应力可能导致大分子的崩溃。此外，还有以省略碳载体，从而消除可能的表面吸收效果，并表明大分子采用在溶液中的构象的可能性。冷冻阴性染色，cryoEM和NS的组合，能提供高对比度的全附件hyd额定试样和也可导致三维重建使用SPA的，但它已不常用的，具有小于10 EMDB条目，这部分是因为污渍本身可与大分子31的完整性干扰。有利于三维重建两个TEM技术是二维电子晶体学和电子断层扫描。 2D电子晶体需要一个平面或管状晶体;晶体的电子衍射用于三维重建可能导致原子分辨率32。在玻璃化的或传统的固定标本的电子断层扫描，大分子或亚细胞组分的透射电镜用于后续断层重建33内旋转，与优点，即单数的对象可以被重建;然而，目前该技术的分辨率极限范围从40至20埃34,35。在所有这些分子TEM技术，对NS和cryoEM数据SPA一直是最广泛的使用。这里说明该协议是专门为获得适合SPA分析cryoEM图像;然而大多数的协议也适用于2D的cryoEM晶体和断层样品。 一个成功的cryoEM会议取决于很多关键步骤合并成功;重要的方面要记住，共同cryoEM文物的解释，以及如何避免它们将在下面的段落描述。本节还介绍了数据收集的指导方针，以获得使用SPA方法高品质的3D重建。 避免过渡到结晶冰。一个主要方面是，样品需要保持在玻璃态从冷冻切入整个TEM观察的时刻。由此切入试样中的液体乙烷后在液氮（-196℃）或液态氦（-269℃）的温度进行的所有后续步骤。升温至高于-135℃的玻璃转化水以结晶水;那么大分子重组可能发生和水结晶称霸图像（ 图3A）;样品应被丢弃。偶然样品预热可能发生，如果冷冻切入，冷冻的网格的容器（即使由gridbox保护），和/或低温保持器插入之间传递到TEM太慢，或者如果处理镊子是不充分的预冷却。在低温时转移的TEM显著真空恶化（冷样品陷阱温暖进气）也可以热身的样本。最后，在辐照的样本也可能导致过渡到结晶冰。 最大限度地减少冰的污染 。由于小样本量（3-5微升）应用到TEM网格，蒸发量可以集中缓冲液成分（盐，洗涤剂），从而影响大分子的完整性，包括多聚态损耗。较高的相对湿度（RH）的微环境或室内规避s此问题。或者冷冻飞泻可以在通风良好的环境冷室来完成。冷冻切入RH后应尽可能地低，以防止冰的污染，或在低温网格环境湿度的冷凝，作为冷冻格本身作为一个冷阱。冰污染由具有高对比度和尺寸范围之间〜5纳米和几微米该干扰甚至完全阻挡图像没有子颗粒。避免空气气流，谈论/对呼吸的空气传输过程中的冷冻格，并降低环境湿度。开液氮容器冷凝水（如白色悬浮颗粒可见）也应被丢弃。 最大限度地提高大分子取向/意见。对于样品的支持，要作出一个选择是真正的多孔格栅和薄碳在多孔网格之间。这取决于（ⅰ）如何将样品遍及碳膜相对于碳的孔，（ⅱ）提供样品concentration，裸孔可以要求的浓度100X比碳载体更高的图像上的类似粒子密度（从〜0.02〜2微米），以及（iii）是如何随机大分子取向的分布。需要注意的是辉光放电，这使得碳的亲水性，将在所有三个方面的重要作用，而这将是样品依赖性。对于大分子的取向，而经常视图是理想的初始结构确定由随机圆锥形重建，炼三维重建到更高的分辨率时的大分子次随机性是可取的。在我们的例子中，RYR1交互与喜爱的"四倍"视图（ 图2D）的碳，需要多孔网格方向的随机性和更高的分辨率36。 最大限度地提高SNR。提高SNR最好的策略是减少噪音的背景来源。过多的冰厚度和（如果存在的话）的碳薄膜的厚度超出所需要的，以支持并嵌入该蛋白质将增加额外的噪声。因此冰厚度应通过控制杂交时间，压力，相对湿度，和滤纸质量被减少到最低限度的必要。对于碳载体，薄（〜5nm）的碳膜层叠在较厚的多孔碳膜:厚多孔碳提供机械阻力而样品被成像在薄碳覆盖的孔。另一方面，请注意，非常薄的冰和/或碳可以导致容易破损支撑，可以变成一个网络（ 图3D）。为了最大化信噪比，应避免任何不必要的缓冲组件。对于膜蛋白，该洗涤剂呈现对比显著收费（ 见图2D，右图）。除了通过减少表面张力的洗涤剂可改变样品散布在载体上的方法。一些膜蛋白需要脂质除了存在下DETergent，这进一步降低了对比度。为了克服这个样本可以之前冷冻暴跌37稀释在缓冲区中低浓度的洗衣粉，没有脂肪。新的替代洗涤剂16并利用纳米盘38，以稳定的跨膜域成分似乎是成功的方法用于成像膜蛋白由cryoEM。 争取高质量的图像和用于CTF校正做准备。上釉标本要求高的散焦值，以产生足够的图像（相位）的对比，其中CTF，即涉及强度与频率的函数，有几个零过渡，在该点没有信息。而CTF校正是没有必要的低分辨率3D重建，旨在为更高的分辨率时，来恢复3D对象的精确表示，具有不同defoci图像需要被收集使得计算CTF校正可以做到的。CTF确定和修正包括在主要的SPA软件包4-7;细节可以在其他地方找到39。为了获得最佳的CTF校正，使用范围defoci的。一个好的策略是交替之间3-4散焦值。的值依赖于该大分子（较大尺寸需要更少的散焦）的大小，冰/碳的厚度（更薄样品需要更少的散焦），并且电压（下限电压需要更少的散焦）。 200千伏，价值观的良好开端范围是2.5，3，3.5和4微米的散焦。周大福表现为交替的高，低强度的戒指（戒指吞40）在倒数空间表示，功率谱。显示所述功率谱将揭示为分辨率的潜力;最宽的可见吞环的频率是接近的可达到的分辨率的先验估计。功率谱应定期检查，并且像散（非圆形）吞环（ 图3C）应与目标象散予以纠正。吞戒指应该是可见至少到所需的分辨率。 使用此协议获得的数据集cryoEM可以直接通过SPA，以产生一个三维重建处理。即使有一个理想的样品中，3D重建的质量将取决于性能和TEM设备的规格，并在图像处理。随着不断改进，在这两个方面，并特别提到了最近开发的直接电子探测器的可能性，以获得三维重建在原子分辨率更一致更接近比以往任何时候。

cryoEM的目标是获得在其天然水合状态大分子的电子显微镜图像。凭借嵌入在玻璃化水的大分子，冷冻水合样品可以直接引入并可视化以TEM仪器。玻璃化，通过闪冻（10,000℃/秒）实现，冻结的样品没有水的结晶，并确保在冻结分子重排是微不足道的。样品的电子散射的相对于周围的缓冲区中的过量提供一种小但足够的对比度，看所述大分子在没有任何污点1。计算机化的图像处理可以被用来重建该大分子的三维结构。大分子的〜500 kDa-多丙二醛的范围是理想的样本cryoEM（代表电子显微镜数据库（EMDB）项的80％以上）;这些包括蛋白质，蛋白质复合物，蛋白质 - 核酸Çomplexes，和膜蛋白包埋在​​双层中。这些大分子是不太可能被结晶（具有小于2％的条目在PDB数据库中），但它是通常的情况是通过x射线晶体学或NMR解决较小的部分可以停靠到由cryoEM创建的3D信封。另一方面，一些由cryoEM解决的最大的结构是由薄截面的TEM 2的全部细胞内识别。因此，有效地cryoEM桥接亚细胞和原子结构之间的尺寸和分辨率的差距。 CryoEM体现更好的大分子的比负染色的更经典方法（NS），其中所述大分子是脱水，由一层重金属染色包围由此染色排除区域产生强烈对比的"负"图像3天然结构。在这两种情况下的电子束产生的大分子的2D投影图像，称为颗粒，其中的SHAPE取决于所述大分子的取向相对于所述电子束。许多颗粒，至少〜1000，并与没有上限，可以在计算机与"单粒子分析"（SPA）软件进行分析，以增加信号到虽然平均信噪比（SNR），并找到粒子的空间取向参数需要重新创建大分子的三维结构由背面投影4-7。 NS，具有更高的SNR，则用于初步样品表征，并产生一个从头三维重建;其第很少超过20埃，这是由重的金属颗粒的脱水和大小强加的。在一般情况下，对于cryoEM三维结构测定，低分辨率3D重建，首先从NS获得然后cryoEM用于细化此低分辨率初始地图进一步研究大分子在其天然水合状态，看它的内部结构，并增加其分辨率。没有德，如果对NS模型被扭曲（最常见的例子是平坦化从脱水8所得）和cryoEM颗粒具有低信噪比，则该失真可以携带到cryoEM模型（模型偏压工件，这是常见的低信噪比数据集9）。结构扭曲会导致的NS和cryoEM原料颗粒之间的原始cryoEM颗粒和3D模型垂直于展平方向的相应凸块之间的不匹配。模型偏差可以自行解决的3D模型，经过连续的优化循环。如果是不发生，这将被检测为缺少来自3D模型生cryoEM颗粒和相应的突起之间的对应关系，然后从头模型应从cryoEM数据构成。一个好的方法可能是使用的角度重构算法10，其可产生几种可能的3D体积，然后用生理盐水三维模型选择最佳的cryoEM模型这将进一步完善11。一个更好的策略是增加的cryoEM数据集的信噪比（参见讨论专用部分）。 纯化大分子的生化质量有最终结果的最大影响。大分子，从组织纯化的，或表达异源系统中，需要具有纯度高，并在结构上不动。它既不应该形成聚集也不应该分解。定义一个特定的构象，制备条件应促进均匀的构象状态。为了研究复合物，它是最佳的那其k个D在nM范围，否则固定剂已被成功地用于稳定低亲和力相互作用12,13。 未处理cryoEM图像上产生的尺寸的有价值的信息，一般轮廓，聚集/多聚化，均匀性的状态，并且macromolecu的内部结构勒。然而该技术的潜力是大大增强了图像处理。的三维结构揭示该大分子的总体架构，配体和亚单位，构象变化的三维位置，并使得能够通过X射线晶体学或NMR测定的原子结构的对接。一个三维重建的信息量逐步增加的分辨率提高:一般15-20一项决议，允许原子结构的明确对接，在9-10α-螺旋如棒可见，在5可以看出β片层，并以3.5埃，更好的分辨率有可能建立的原子模型14。 的可能性，以获得翔实的图片，并使用SPA从相对少量的样品做出cryoEM一个有吸引力的技术，三维重建，3-5微升至少为0.05毫克/毫升就足够准备一份TEM网格。最低要求的工具对于cryoEM是自制或商业冷冻柱塞，具有超高真空，图像记录介质和低剂量的试剂盒，一个低温保持器与它的泵站，辉光放电装置和蒸发器的电子显微镜。在TEM非必要但进一步需要的特征是CCD照相机（存在于所有的现代电信设备制造商）或直接电子检测器，场发射枪（FEG），能量过滤和高吞吐量数据收集软件。这个软件在原则使得能够收集颗粒的数以万计在单个cryoEM会话15，但是增加了数据存储需求和随后的监督后处理时间需要的要加以考虑。 FEG结合对比度传递函数（CTF）修正影响那些达到的分辨率足以显现α-螺旋大多数3D重建。在这一点上，分辨率的高低也是样品依赖性:膜蛋白达到10的分辨率是不常见的，而如果对称高阶的存在，那么原子分辨率更可以实现的。直接电子检测器的最近发展已使原子分辨率即使对于大分子低（4次）或不对称，其中，从X射线衍射数据16,17，这些衍生的cryoEM电子密度图的质量相匹配。 提供说明了该技术的能力的实际例子在这里的四个重要类型的大分子，其中cryoEM在其结构决定一个持续主要作用。 （一）在他们的二十面体对称，增加了60倍，其大尺寸允许忠实的 ​​和可重复的定位数据集，几个二十面体病毒的结构已经解决了由cryoEM近原子分辨率其次SPA 18。我们展示了一类的二十面体病毒，腺相关病毒（自动增值服务）的一个例子，对于其通过cryoEM和由cryoEM / X射线HY近原子结构布里德方法存在19,20。 （ⅱ）大分子与螺旋结构包括微管，微丝和病毒。其重复单元沿着螺旋轴可以由更复杂的版本的SPA适于将螺旋几何形状21进行分析。在这个例子中，我们表明烟草花叶病毒（TMV），已被解决，以原子级分辨率由cryoEM 22的RNA病毒。 （三）大的可溶性蛋白被充分研究。其中，大分子形成对称多聚缸构成经常性的架构往往在解决亚纳米分辨率23,24。这里，我们表明，KLH，无脊椎动物氧载体，其中混合的分子模型，从cryoEM / X射线晶体混合方法25中创建的图像。 （ⅳ）膜蛋白是蛋白质的一类重要的;它们构成的约1/3由人类基因组编码的蛋白质的，但它们难以表征结构由于与TR相关的复杂性ansmembrane域稳定26，构成由任何结构相结合的技术解决了这个结构的小于1.5％。 cryoEM及三维重建在其结构测定的作用例举与兰尼碱受体（RyR的），一个大的真核细胞内Ca 2+通道在肌肉收缩和脑的信号非常重要。最高分辨率cryoEM结构到今天为止，有10的分辨率，显示二级结构27。

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Name of Material/ Equipment</td>
			<td style="width:132px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:243px;">Ethane</td>
			<td style="width:132px;">Airgas</td>
			<td align="right" style="width:119px;">371745</td>
			<td style="width:167px;">99.99% purity very important, impurities can lead to high-contrast contaminants on the grid. CAUTION: flammable</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:243px;">Liquid nitrogen</td>
			<td style="width:132px;">Airgas</td>
			<td align="right" style="width:119px;">27135</td>
			<td style="width:167px;">99% purity. CAUTION: handling liquid nitrogen in a cold room poses a serious asphyxia hazard since nitrogen displaces oxygen without any warning signs.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont no. 5 Antimagnetic tweezers</td>
			<td style="width:132px;">Ted Pella</td>
			<td align="right" style="width:119px;">5326</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Mica sheets, 1 x 4 cm</td>
			<td style="width:132px;">Ted Pella</td>
			<td align="right" style="width:119px;">54</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:243px;">Graphite rods, presharpened size 1/8&#8221; dia x 2 &#188;&#8221; long tip 0.039&#8221; dia neck type</td>
			<td style="width:132px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">70220-45</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:243px;">350 ml cryo-dewars</td>
			<td style="width:132px;">Pope</td>
			<td align="right" style="width:119px;">8600</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:243px;">Cu 400 mesh holey grids&#160; Quantifoil</td>
			<td style="width:132px;">Quantifoil</td>
			<td style="width:119px;">Q10525</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:243px;">Cu 400 mesh holey grids C-Flat</td>
			<td style="width:132px;">Protochips</td>
			<td style="width:119px;">CF-1.2/1.3-4C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:243px;">Glow discharge device</td>
			<td style="width:132px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">Model E7620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:243px;">Turbo pumping station</td>
			<td style="width:132px;">Gatan</td>
			<td style="width:119px;">Model 655</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:243px;">Carbon evaporator</td>
			<td style="width:132px;">Denton Vaccum</td>
			<td style="width:119px;">Model 502B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:243px;">Freeze plunger</td>
			<td style="width:132px;">FEI</td>
			<td style="width:119px;">Vitrobot Mark IV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:243px;">Image Processing software CTFFIND3/CTFTILT</td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://grigoriefflab.janelia.org/ctf</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:243px;">Image Processing software EMAN</td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://blake.bcm.edu/emanwiki/EMAN2</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:243px;">Image Processing software Spider</td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://spider.wadsworth.org/spider_doc/spider/docs/spider.html</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:243px;">Image Processing software Freealign&#160;</td>
			<td style="width:132px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://grigoriefflab.janelia.org/frealign</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">3D visualization software Chimera</td>
			<td></td>
			<td></td>
			<td style="width:167px;">http://www.cgl.ucsf.edu/chimera/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Native Keyhole Limpet Hemocyanin</td>
			<td style="width:132px;">Biosyn</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

do's and don'ts of cryo-electron microscopy: a primer on sample preparation and high quality data collection for macromolecular 3d reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

小心执行所有在图1中概述的协议步骤的使冷冻水合，非染色大分子的可视化。显示结果为四个具有代表性的试样不同的结构特征。由NS和cryoEM的方法获得的它们的显微图像进行比较（ 图2）。 AAV5（ⅰ）的NS图像揭示了大约130的直径为病毒样颗粒。充分和空结构并存的对应破损（允许进入染色）和结构完整的衣壳，分别。 CryoEM显示均匀空结构;的确，这些病毒颗粒不含有遗传物质28。 NS显示主要的圆形颗粒，而cryoEM显示大部分六角形颗粒，反映了他们的二十面体的形式。 （二）180直径和长度可变螺旋TMV秀棒的照片，往往长于0.1微米，具有23可见螺旋重复这两个在NS和cryoEM。在40埃中心通道填充有污点的NS图像和空中cryoEM图像。 （三）本机KLH，8丙二醛一个didecamer，装配在350直径和400的长度特征万桶。 NS和cryoEM显示KLH的矩形方的意见和循环最终的意见。这两种技术都揭示了6条纹垂直于所述对称轴和每层内的等距形态学单元。 CryoEM揭示了一个空的内部结构。 （四）兰尼碱受体，2.26丙二醛的大型四聚体离子通道，被看作是275 X 275 2方。复杂的表面特征，如中央十字和中心凹陷表现为色斑积累NS，并通过cryoEM密度较低的地区。虽然NS显示所有四个测试样品中类似的对比，所述cryoEM板比较暴露RYR1的低信噪比（对比度）由于洗涤剂的存在下，以溶解该膜蛋白。 cryoEM工件的典型例子示于RYR1:结晶冰，冰的污染，像散及幅状结构（ 图3）。其原因与预防讨论的章节详细描述。 SPA及三维重建冷冻水合RYR1揭示其四倍对称结构，这反映了蛋白的同源四聚体组织（ 图4A）。胞质域构成的275 X 275×120埃3方形棱镜和包含多个可再现的球状结构域形成的支架状结构。跨膜结构域形成了一个小的锥形方形棱镜最大尺寸115 X 115 X 60 3。 10的分辨率，可以可视化α-螺旋（ 图4B）。三维差映射可以揭示重要配体的位置，在这种情况下FKBP12的三维差图，12 kD的蛋白质求索RYR1红色的亚基，是叠加在RYR1（ 图4C）29。最后，构象变化提供大分子的动态视图。在这种情况下，RYR1在关闭或打开的条件预先稳定化揭示了一个质量易位从闭合到打开状态（ 图4D）27。 <img alt="图1" src="/files/ftp_upload/52311/52311fig1.jpg" /> 在低温电子显微镜技术图1.流程图。该图突出了协议的主要步骤。 <img alt="图2" src="/files/ftp_upload/52311/52311fig2.jpg" /> 图对各种样品2对比NS和cryoEM的。 （A）AAV5，一个二十面体病毒。（B）TMV，螺旋病毒。插图显示该螺旋重复的23埃，（C）的本机KLH（D）RYR1;代表性的观点是强调了（F，四倍观点和s，侧视图）。所有样品都在低剂量条件下和在50,000X一个下为200千伏的加速电压相同的放大倍率成像。比例尺，10纳米。所有NS样品在碳载体，cryoEM样品A，C，D都是薄的碳过的Quantifoil和cryoEM样品B是直接通过的Quantifoil孔。 <a href="https://www.jove.com/files/ftp_upload/52311/52311fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52311/52311fig3.jpg" /> 图3.图库图像显示上RYR1 cryoEM样本常见cryoEM文物。 （A）结晶冰。（B）冰污染（箭头）。（C）散光。在像散像RyR1s都隐约可见。右边的插图显示了电源SPECT朗姆酒不对称吞戒指的形象。左边的插图显示，以供参考非像散像的功率谱。（D）网络状结构。 <a href="https://www.jove.com/files/ftp_upload/52311/52311fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52311/52311fig4.jpg" /> 图4. CryoEM和三维重建兰尼碱受体。 （A）的等值面表示（B）的 RYR1的N-末端30的晶体结构示出的原子坐标和cryoEM信封之间良好的一致性的对接;箭头指向两个α螺旋，从该结构伸出。箭头表示的四个螺旋环绕离子通道的孔中的一个。虚线表示该膜的边界。（C）的 RYR1和3D LO在FKBP12配体（橙色）的阳离子。（D）RYR1在从封闭（橙色）要开（黑目）构构象变化。 <a href="https://www.jove.com/files/ftp_upload/52311/52311fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction at JoVE.com

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

该做什么和不该做什么低温电子显微镜的:入门样品制备和高质量的数据采集为大分子三维重建

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

do-s-don-ts-cryo-electron-microscopy-primer-on-sample-preparation

Research

JoVE Journal

Biology

45.9K 次观看.  Virginia Commonwealth University. This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

视频: Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

科研

生物学

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列:样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

剖检诊断，并选择在大鼠和小鼠的组织和样品收集

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

编制质量肌醇Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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 ...