Name: tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy
Uploaded: 2014-10-13T14:00:00.000+00:00
Duration: 12 min 52 s
Description: Watch this Scientific Journal Video about Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy at JoVE.com

加州大学伯克利分校的肯特麦当劳博士的善良和慷慨是极大的赞赏。我们感谢匿名审稿人非常有帮助的建议。伯奇 - 史密斯的实验室正在启动从田​​纳西大学的​​资金支持。

注:QFS过程需要格外小心和谨慎的用户，我们强调这些安全注意事项这里的注意事项和注意事项（如适用）。 1.Preparation的HPF运行<ol><li>在开始样品制备，打开高压冷冻以下制造商的说明。 注:在本协议中所使用的高通滤波器单元是Wohlwend紧凑型02单位（ 图1A），它大约需要一点差一分，和一个半小时的启动程序运行高通滤波器可以开始之前。 <ol><li>开关上的Wohlwend紧凑型02高压冷冻。 <ol><li>通过旋转从"0"主开关"1"打开仪器。 </li><li>释放压缩空气的机器。 </li><li>释放液氮到机器。 </li><li>按"系统干起来"按钮。 </li><li> 30分钟后，按"SYSTEM枯竭"了按钮。 </li><li>按下"ON / OFF"按钮，在仪器上进行切换。 </li><li>按"氮"按钮。 </li><li>让机器冷却20分钟，即使在"驱动器"按钮已经亮了起来。 </li><li> "驱动器"按钮会亮起。 </li><li>按"驱动器"按钮。 </li><li>按下"自动"按钮。 </li><li> "系统准备就绪"按钮会亮起。 </li><li>将温度和压力探头放入压力室，并与针将其锁定。 </li><li>按"JET AUTO"按钮。该步骤检查，该压力增加和温度的降低是所希望;本质上是一个试运行。急剧下降的温度和大幅增加的压力，预计（ 图1B）。 </li><li>进行两次以上的JET周期。 </li></ol></li></ol></li></ol> 2，准备Receivi吴冷冻样本<ol><li>填充含有用液氮冷冻管座的绝缘框（参见安全警告），以使支架完全覆盖（ 图1C）。 注意:使用个人防护装备，包括cryogloves和护目镜液氮操作时。 </li><li>放置装有将FS介质插入在液氮中（ 图1C）的铝管座的小瓶的适当数目。与此协议多达四个盘各自含有一个单一的样品可以被放置在一个单一的离心管。 注:该瓶应标有锐器像钻石尖书记和非常柔软的铅笔。 <ol><li>对于QFS在固定液中丙酮9用1％OSO 4和0.1％醋酸铀。准备在大体积的溶液，取1.5毫升分装到冷冻管并储存在液氮中冷冻。 注意:遵守安全警告处理OSO 4和醋酸铀。 注:OSO 4 </suB&gt;和醋酸铀的危险化学品的，应在通风橱中进行处理，而穿着适当的个人防护装备（PPE），包括封闭趾鞋，白大褂和手套。 请注意:用于保持样品QFS的冷冻管应具有硬的O形环，以确保所述管中QFS保持密封，以避免OSO 4的泄漏。 </li></ol></li><li>通过混合面包酵母用10％的甲醇，用牙签或其他类似工具大致相等音量，直至糊状平滑准备酵母膏。酵母膏作为一种胞外冷冻保护剂和用于填充在试样载体样品周围的空间。糊的量取决于样品的数目; 10个样品或更少的膏（1ml）中，可以混合于微量离心管中。 </li></ol>样品3的高压冷冻<ol><li>从植物中取出的利息叶（或其他组织），并轻轻将它放在一块登塔的升蜡或其它切割表面。使用2.0毫米或所需的大小冲床切割样品出来的叶子。用钳子轻轻地处理样品，并尽快制定越好。 </li><li>在解剖显微镜下工作，将叶盘在A型标本载体的0.2mm的侧面和酵母膏完全覆盖。确保在盘被完全填充并且膏是用保持器的通过提供平滑的糊状物 ​​用细画笔（ 图2）的边缘的水平。 </li><li>放置在试样保持器的载流子。支付与B型试样载体，表面平坦下来的样本。 注:该试样夹持器应是干燥的，并在室温下进行。 </li><li>取样品中试样夹持器，将其插入到机器，并按下"JET AUTO"按钮，它应该完成一两秒钟启动冷冻循环。 </li><li>尽可能快地工作，从机器中取出支架，然后将尖端拿着SAMP勒入液氮中的HPF机顶部的绝缘箱。浸泡两对钳子的前端，在液氮来冷却它们。 注:冻结后，运营商只能用液氮冷却的镊子处理。暖（室温）下钳通常是失败的cryotechniques的原因。 </li><li>打开包含在FS介质离心管，然后将盖在盒子的侧面。有一对液氮冷却的镊子的辅助下，轻轻地从试样保持器中取出光盘，确保光盘总是在液氮或蒸气。工作在液氮蒸气，保持在FS管与一个预冷却的钳子，使用其他的支架放置在FS小瓶。拧FS小瓶的盖子重新打开。不要捕获任何液氮瓶中。 注意:当样品转移到离心管的QFS，确保没有液氮被困在小瓶。液氮变暖和任何霉期间扩大700倍ð液氮可以在这段时间内会引起爆炸。 </li><li>重复步骤3.1至3.7，直到所有需要的样本被冻结。包含相同类型的样品的多个叶盘可放置在相同的小瓶中。另外，试样夹持器需要被干燥并升至室温冷冻运行之间。要快速执行此操作，用吹风机加热，并监控其温度通过触摸。 </li></ol> 4，准备冻结换人<ol><li>完全浸没在液氮中的铝加热块为10分钟或直至核沸腾停止。而这会上，放置一个层干冰（1-2厘米）用作FS腔（ 图3），该容器的底部。甲泡沫聚苯乙烯容器或冰桶可用于FS，随着碎干冰或粒料。 </li><li>快速插入含有样品随温度探头插入中间行加热块的离心管。确保在盖子小瓶紧紧拧上，使得FS介质FS的过程中不会漏出。开始记录温度。 <ol><li>通过用离心管的顶部放置一个热电偶，使其到达管底使温度探测器。密封该管的盖用树脂使得没有液体泄漏出去。填用1.5ml丙酮的管在每个QFS运行的开始。 </li></ol></li><li>使用绝缘cryogloves或大钳，倒出来从加热器块中的所有液态氮。请注意不要倒出来的冷冻管。 注意:使用个人防护装备，包括cryogloves和护目镜液氮操作时。 </li><li>放置装有将小瓶上的干冰中QFS室内的层块。确保该块被放置以使管子躺在水平，但有轻微的向上翘起。不应有渗漏，如果正确的离心管中使用。 </li><li>打包QFS室用干冰使FS管覆盖，虽然它是没有必要覆盖块的顶部。放置于所述腔室的盖子。 </li></ol> 5，快速的FS 注意:在OSO 4的任何泄漏发生在不经意间，尽管其他的注意事项时通风柜执行QFS运行。 <ol><li>将QFS室在一个平台上的旋转振动筛在通风橱中，并旋转125转120分钟。在此期间，该块的温度应逐渐增加至约-80℃。搅拌，确保各组分混合为更好的FS。 注:在摇床中通风柜和通风柜门的位置的位置应在整个QFS程序和运行，以确保样品的一致变暖每QFS相同。 </li><li>除去从腔室中的干冰，并继续振荡培养1小时。 注意:温度应提高到约-15℃至-20℃。 </li><li>从QFS室中取出样品，温度探头和放置在摇床上于室温下再10-15分钟，直至它们达到室温。停止记录时的温度。 </li><li>在财政司司长，关闭HPF机。 <ol><li>关闭液氮罐的抽头而将HPF机被充入氮气。 </li><li>按"氮"按钮。 </li><li>等到红色的"活塞下行"光熄灭。 </li><li>按"开/关"按钮。 </li><li>按"系统干起来"按钮。 </li><li>切断压缩空气到本机的供应。 </li><li>至少12小时后（以确保任何湿气的干燥），通过接通主开关从"1"到"0"关掉机器。 </li></ol></li></ol> 6财政司司长后处理<ol><li>小心在适当的容器有毒废物移除媒体的FS​​用塑料移液管和地点。 注:OSO 4和醋酸铀的危险化学品并应在通风橱中，而穿着适当的个人防护装备（PPE），包括封闭趾鞋，白大褂和手套进行处理。 </li><li>洗涤样品四次用100％丙酮。收藏前两个洗涤，并放置在有毒废物容器。 </li><li>用细镊子，从人取出的组织样本。保持湿润​​的样品用丙酮为做到这一点，而且工作非常轻柔，以免破坏样品。 注意:这是不寻常，它实际上可能是有酵母膏落入远离样品在这一点上是有用的。酵母膏通常是非常深褐色，而叶组织仍然是绿色的。 </li><li>收集的样品中含有丙酮离心管。按照通常的协议与样品制备（渗透和嵌入）的透射电子显微镜进行。 </li></ol>

<ol>
	<li>Studer, D., Humbel, B. M., Chiquet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+high+pressure+frozen+samples+bridging+the+gap+between+cellular+ultrastructure+and+atomic+resolution.">Electron microscopy of high pressure frozen samples bridging the gap between cellular ultrastructure and atomic resolution.</a> Histochemistry and cell biology. 130, 877-889 (2008).</li><li>Studer, D., Hennecke, H., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+of+soybean+nodules+leads+to+an+improved+preservation+of+ultrastructure.">High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure.</a> Planta. 188, 155-163 (1992).</li><li>Bowers, B. a. M. M., Crang, R. F. E., Klomparens, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+2.">Ch 2.</a> Artifacts in Biological Electron Microscopy. 2, 13-42 (1988).</li><li>Mollenhauer, H. H., Crang, R. F. E., Klomparens, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+3.">Ch 3.</a> Artifacts in Biological Electron Microscopy. , 43-64 (1988).</li><li>Kiss, J. Z., Giddings, T. H., Staehelin, L. A., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ultrastructure+of+conventionally+fixed+and+high+pressure+frozen/freeze+substituted+root+tips+of+Nicotiana+and+Arabidopsis.">Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis.</a> Protoplasma. 157, 64-74 (1990).</li><li>Moor, H., Steinbrecht, R. A., Zierold, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cryotechniques in Biological Electron Microscopy. , 175-191 (1987).</li><li>Vanhecke, D., Graber, W., Studer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close-to-native+ultrastructural+preservation+by+high+pressure+freezing.">Close-to-native ultrastructural preservation by high pressure freezing.</a> Methods in cell biology. 88, 151-164 (2008).</li><li>Dahl, R., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+the+preservation+of+biological+structure+theory+and+practice.">High-pressure freezing for the preservation of biological structure theory and practice.</a> Journal of electron microscopy. 13, 165-174 (1989).</li><li>McDonald, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+preservation+of+high+resolution+fine+structure+and+antigenicity+for+immunolabeling.">High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling.</a> Methods in molecular biology. 117, 77-97 (1999).</li><li>Hippe-Sanwald, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freeze+substitution+on+biological+electron+microscopy.">Impact of freeze substitution on biological electron microscopy.</a> Microscopy research and technique. 24, 400-422 (1993).</li><li>Kiss, J. Z., McDonald, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+immunocytochemistry+following+cryofixation+and+freeze+substitution.">Electron microscopy immunocytochemistry following cryofixation and freeze substitution.</a> Methods in cell biology. 37, 311-341 (1993).</li><li>Segui-Simarro, J. M., Austin 2nd, J. R., White, E. A., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+tomographic+analysis+of+somatic+cell+plate+formation+in+meristematic+cells+of+Arabidopsis+preserved+by+high-pressure+freezing.">Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by high-pressure freezing.</a> The Plant cell. 16, 836-856 (2004).</li><li>Kobayashi, K., Otegui, M. S., Krishnakumar, S., Mindrinos, M., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INCREASED+SIZE+EXCLUSION+LIMIT+encodes+a+putative+DEVH+box+RNA+helicase+involved+in+plasmodesmata+function+during+Arabidopsis+embryogenesis.">INCREASED SIZE EXCLUSION LIMIT encodes a putative DEVH box RNA helicase involved in plasmodesmata function during Arabidopsis embryogenesis.</a> The Plant cell. 19, 1885-1897 (2007).</li><li>Austin, J. R., 2nd, L. A., Staehelin, <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+architecture+of+grana+and+stroma+thylakoids+of+higher+plants+as+determined+by+electron+tomography.">Three-dimensional architecture of grana and stroma thylakoids of higher plants as determined by electron tomography.</a> Plant physiology. 155, 1601-1611 (2011).</li><li>McDonald, K. L., Webb, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze+substitution+in+3+hours+or+less.">Freeze substitution in 3 hours or less.</a> Journal of microscopy. 243, 227-233 (2011).</li><li>Taiz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plant+Vacuole.">The Plant Vacuole.</a> The Journal of experimental biology. , 172-1113 (1992).</li><li>Wilson, S. M., Bacic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+plant+cells+for+transmission+electron+microscopy+to+optimize+immunogold+labeling+of+carbohydrate+and+protein+epitopes.">Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes.</a> Nature protocols. 7, 1716-1727 (2012).</li><li>Ding, B., Turgeon, R., Parthasarathy, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substructure+of+freeze-substituted+plasmodesmata.">Substructure of freeze-substituted plasmodesmata.</a> Protoplasma. 169, 28-41 (1992).</li><li>McDonald, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Embedding+Methods+into+Epoxy+and+LR+White+Resins+for+Morphological+and+Immunological+Analysis+of+Cryofixed+Biological+Specimens.">Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens.</a> Microscopy and microanalysis. 20, 152-163 (2014).</li><li>McDonald, K. L., Auer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing,+cellular+tomography,+and+structural+cell+biology.">High-pressure freezing, cellular tomography, and structural cell biology.</a> BioTechniques. 41, 137, 139-141 (2006).</li><li>Spiegelhalter, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+dynamic+live+cell+imaging+to+3D+ultrastructure+novel+integrated+methods+for+high+pressure+freezing+and+correlative+light-electron+microscopy.">From dynamic live cell imaging to 3D ultrastructure novel integrated methods for high pressure freezing and correlative light-electron microscopy.</a> PloS one. 5, e9014 (2010).</li><li>McDonald, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+high-pressure+freezing+preparation+techniques+for+correlative+light+and+electron+microscopy+of+the+same+cells+and+tissues.">A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues.</a> Journal of microscopy. 235, 273-281 (2009).</li></ol>

The authors have nothing to disclose.

这里介绍的协议的成功在很大程度上依赖于该用户。首先，需要提前准备，以确保所有必要的材料都是现成的，并以足够的量来完成整个HPF-QFS运行。第二，用户必须迅速，从步骤到步骤移动以最小化的样品处理以有效的方式，从而减少改变的组织的天然状态。一旦样品被冻结，他们是脱水之前就必须让它们保持低温，所以必须小心，以避免任何处理，可以在不经意间升温的样品，例如，处理一个...

我们的细胞超微结构的知识主要来源于电子显微镜，它可以在几纳米1的范围内解决的细节。尽管是如此强大，在透射电子显微镜的分辨率不考虑人性化，样品制备，需要费时费力的协议，并从医生需要一定的专业知识。样品的传统固定结合了使用的醛和四氧化锇在进一步处理之前，包括脱水，在树脂包埋，然后切片，以产生超薄切片被，然后用重金属染色。然而，已知的是化学固定可产生伪影，包括蛋白质聚集和脂质1的损失，并以膜，最终影响多种细胞区室2的变化。这些构件在很大程度上归因于固定和脱水的缓慢速率在 ​​室温下3，4，5。 6,7。HPF减小Ä样品的温度至该液态氮的，非常高的压力（210兆帕或2100巴）的以毫秒为单位下。如果处理得当HPF防止形成大的冰晶，能导致细胞超微结构的重大伤害。 HPF可用于固定的100-200微米厚的样品在生物溶质7的典型浓度。上有物理多次审查和原则基础HPF， 如 1，7，8。 HPF后，将样品在低温下（-78.5℃培养至-90液体含有机溶剂的化学固定剂如四氧化锇，一般几天的存在C），＃176。在这样低的温度下，水的样品中的被替换的有机溶剂，通常是丙酮，甲醇1,9。因此，该过程被称为冷冻替代（FS）。然后将样品逐渐加热，并在这段时间内是固定的，通常用四氧化锇和乙酸双氧铀9。交联在低温下具有固定的被固定1分子的优点。 FS因此产生的优良品质相比，那些在室温下固定用常规的化学固定，特别是它导致改进的超微结构保存，保存较好的抗原的样品，并减少未结合的蜂窝部件10，11的损失。 最FS进行过长期的时间段，通常长达数天。这是特别TRUE对于植物样品12，13，14。麦当劳和韦伯开发的最新协议，大大降低了时间的FS从几天到几小时15。在他们的快速冻结取代（QFS）过程，FS进行超过3小时，而在超级快速的FS（SQFS）样品在90分钟内进行处理。通过这些方法产生的样品的质量比得上那些用传统的FS协议产生。我们已经通过了QFS协议植物样品的高通滤波器后的下游加工。这已被证明不仅节省时间，而且钱，QFS和SQFS使用，而不是昂贵的市售FS机常见的实验室设备。 植物组织往往是非常具有挑战性的透射电子显微镜做准备。平均来说，植物细胞是大于或者细菌或动物细胞。疏水性蜡质角质层厚的细胞壁，含有有机酸，水解酶和酚类ç大型充水空泡的存在ompounds可以占据多达90％的总细胞体积16，并且充气空间的存在严重降低了系统17的热传导性。另外，在植物的情况下，样品的厚度几乎总是超过20μm时，限制使用化学固定的。在这些厚度中，水的导热系数低的防止冻结速率大于约10,000℃/秒，在样品的中心。这个速度是必需的，以避免破坏性的六角形冰的形成（冰晶具有较低的密度大于10〜15纳米）8。总之，这些挑战存在于样品和随后的FS既适当冻结。尽管如此，冷冻固定是固定植物样品的最好方法。在这里，一个协议，用于HPF-QFS植物组织样本呈现。它集中于模型物种拟南芥 ，但也被用于与本氏烟草 。典型的结果表明，HPF-QFS产生SA同等质量的传统HPF-FS中的一小部分时间mples。用适当的调整，该协议也可以被用于其他相对较厚的生物样品。

<table><tbody><tr><td>Wohlwend HPF Compact 02 High Pressure Freezing Machine</td><td>Technotrade International, Inc</td><td>HPF02</td><td>With integrated oscilloscope to display freezing and pressure curves; PC (not included) is required for display&amp;nbsp; of freezing parameters</td></tr><tr><td>Holder for DN 3 x 0.5 mm aluminum apecimen carriers</td><td>Technotrade International, Inc</td><td>290</td><td></td></tr><tr><td>Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type A</td><td>Technotrade International, Inc</td><td>241-200</td><td></td></tr><tr><td>Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type B</td><td>Technotrade International, Inc</td><td>242-200</td><td></td></tr><tr><td>Storage Dewar 20.5 L, MVE Millennium 2000 XC20</td><td>Chart</td><td></td><td></td></tr><tr><td>Baker&#039;s yeast</td><td></td><td></td><td>The older the better, to avoid excessive gas (CO&lt;sub&gt;2&lt;/sub&gt;) production</td></tr><tr><td>Tooth picks</td><td></td><td></td><td></td></tr><tr><td>Thermocouple data logger EL-USB-TC</td><td>OMEGA Engineering Inc.</td><td>OM-EL-USB-TC&amp;nbsp;</td><td>Replacement battery purchased separately</td></tr><tr><td>Temperature probe</td><td>Electron Microscopy Sciences</td><td>34505</td><td></td></tr><tr><td>Heater block 12/13 mm</td><td></td><td></td><td></td></tr><tr><td>Rotary shaker</td><td>Fisher Scientific</td><td>11-402-10</td><td></td></tr><tr><td>Leaf punch - Harris Uni-core 2.00</td><td>Ted-Pella Inc.</td><td>15076</td><td></td></tr><tr><td>Pink dental wax</td><td>Electron Microscopy Sciences</td><td>72660</td><td></td></tr><tr><td>Cryogenic vials 2 ml</td><td>Electron Microscopy Sciences</td><td>61802-02</td><td></td></tr><tr><td>Methanol</td><td></td><td></td><td></td></tr><tr><td>Blow dryer</td><td></td><td></td><td></td></tr><tr><td>Dry ice</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td>Several pairs</td></tr></tbody></table>

tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy

自从20世纪40年代的透射电子显微镜（TEM）一直提供生物学家与生物材料的超高分辨率图像。然而，因为费力和费时的协议也需要经验中制备无伪影的样品的，TEM不被认为是用户友好的技术。对于传统的TEM样品制备中使用的化学固定剂保存细胞结构。高压冷冻是生物样品在高压下的冷冻固定，以产生非常快的冷却速率，从而限制了冰的形成，这是不利于细胞的超微结构的完整性。高压冷冻和冷冻替代目前所选择的用于制造高质量的形态在用于TEM树脂部分中的方法。这些方法最大限度地减少通常与传统的加工相关的超薄切片透射电镜的文物。冷冻固定后的冷冻水的样品中被替换为液体在低温条件下的有机溶剂中，这个过程被称为冷冻替代。冷冻替代典型的是在上专用的，昂贵的设备数天。最近的创新允许而不是通常的两天过程在三小时内完成。这之后通常是样品制备，包括浸润和切片之前在环氧树脂包埋的几天。在这里，我们提出了一个协议，结合高压冷冻和速冻置换，使植物样品固定待小时内完成。该协议可以很容易地适于与其他组织或生物体的工作。植物组织是因为加气空间和装满水的空泡阻碍水无冰冷冻的存在，特别令人关注。此外，化学固定的方法是在由于细胞壁阻碍的化学品渗透至深处的组织内的植物特别长。植物组织，因此particularly具有挑战性的，但这个协议是可靠的，并产生最高质量的样品。

结果下面介绍使用Wohlwend紧凑型02 HPF（ 图1A）已经获得。这种仪器的一个主要优点是简单易用的标本运营商和持有人的。当使用其他乐器，麦当劳建议两个用户应进行样品制备和HPF，人们准备的样品，而其他则冻结并转移到FS的冷冻管9。然而，Wohlwend标本运营商和持有人是很容易为单个用户独立操作（ 图2A，B，E，F和G）。然而，应当指出，应该进行一些试运行了?...

Watch this Scientific Journal Video about Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy at JoVE.com

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

tandem-high-pressure-freezing-quick-freeze-substitution-plant-tissues

Research

JoVE Journal

Biology

16.3K 次观看.  University of Tennessee, Knoxville. Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

视频: Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

硅氮化物膜的细胞培养和同步加速器X射线荧光纳米分析的冷冻制备

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

科研

生物化学

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

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 ...

生物学

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

通过低温扫描电子显微镜研究光合膜内冻结，割断叶片组织的超分子组织

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

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 ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

闪存和冻结:一种新的技术来捕获膜动力学与电子显微镜

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

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 ...