Name: plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy
Uploaded: 2017-05-05T12:30:00.000+00:00
Duration: 13 min 35 s
Description: Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

我们对此表示感谢M. Bouchecareilh，E.Tétaud，S. Duvezin-Caubet和A.德文的帮助和意见的手稿。我们非常感谢何地拍摄的图像波尔多成像中心的电子成像极。这项工作是由中心法国国家科学研究的支持。

1.福尔瓦网格薄膜的制备注意:在使用个人防护设备（手套，实验室外套，眼镜）在通风橱下执行氯仿操纵。使用400个目电子显微镜铜网格。与其他类型的网格（其它的网孔尺寸，金和镍网格），冷冻的质量较差。 <ol><li>制备0.3％的聚乙烯醇缩甲醛的氯仿100mL的溶液。允许不搅拌溶解过夜。 </li><li>把400目在玻璃小瓶中电子microscopycopper网格（直径3.05毫米）（高度为3厘米，直径为2厘米）含丙酮（每平方英寸的孔数）。搅拌与手的圆形运动的玻璃小瓶中。用塑料移液管除去丙酮。允许溶剂蒸发5分钟。通过倾倒它们放到滤纸转移网格和使其干燥5分钟。 </li><li>通过用无绒布直到发亮/滑擦拭抛光76×26 平方毫米的载玻片。</li><li>取结晶（直径15厘米，高度:7厘米，容量1200mL）中，并将其填充到用蒸馏水的边缘。通过使玻璃棒表面上两次以除去灰尘清洁水表面。 </li><li>保持两个手指之间的载玻片上并在几秒钟的聚乙烯醇缩甲醛溶液蘸。拿着它直立的倒杯下干燥5分钟。 </li><li>当膜是干的，得分载玻片的边缘用锋利的剃刀刀片以限定膜的极限，以从滑动地移位。 </li><li> 深吸一口气，呼气巨资到幻灯片上和电影渲染影片稍微有点不透明，提升其知名度得到下水蒸气层。 <ol><li>由具有大约30°的角度接触滑动的底部立即浮在薄膜上结晶水的表面。让从滑动膜释放和剥开到结晶器的水表面。轻轻一拉片脱落与镊子，如果需要的话。 </li></ol></li><li>轻轻地将栅格面朝下放在膜漂浮在水面上是适当的厚度（约10nm）和没有皱纹的区域。 </li><li> 通过覆盖与约瑟夫纸栅格拿起膜作为它们漂浮在结晶器水面上。 <ol><li>保持的约瑟夫纸用一只手，触摸约瑟夫纸的一端到膜的一端。等到约瑟夫纸是完全湿透。与粘在网格中取出约瑟夫纸。 </li></ol></li><li>放置网格用于在覆盖的培养皿中进行最后干燥并在室温下在使用前储存24个小时。 </li></ol> 2.冻结取代介质的制备注:将四氧化锇（OSO 4）和醋酸铀是危险化学品。处理它们在通风橱而穿着适当的个人防护设备（PPE），其包括实验室外套和手套。 FOLLOW处理的安全警告（OSO 4）和乙酸双氧铀。使用O形环的冷冻管，以避免的OsO 4的泄漏。 <ol><li> 超微结构研究 <ol><li>把玻璃的OsO 4安瓿在玻璃烧瓶中。 </li><li>通过晃动瓶打破安瓿。 </li><li>添加丙酮的适当体积的100％，得到4％的最终浓度。 </li><li>搅拌和分配1.5毫升等分试样到1.8毫升离心管。 </li></ol></li><li> 蛋白质免疫标记 <ol><li>放置0.05克乙酸双氧铀的在玻璃烧瓶中。 </li><li>加入50mL 100％丙酮中。 </li><li>搅拌用磁力搅拌器将溶液。当溶液变得澄清，用0.2μm过滤器过滤。 </li><li>分配1.5毫升分装成1.8毫升离心管。 注:事先准备含有冷冻置换介质的冷冻管的切入冷冻过程，并保存在-84℃冷冻机中。反转玻璃烧瓶扔掉4的OsO安瓿的危险废物。 </li></ol></li></ol> 3.细胞的制备注:此步骤是该方法成功的关键。对于所有类型的细胞，培养细胞获得细胞的数量表示。离心机中在指定的时间和速度指示管。 <ol><li>为了获得从不同的细胞类型的颗粒的适当的一致性，生长的细胞如下: <ol><li> 酿酒酵母和粟酒裂殖酵母 ，在5mL最小或完全液体介质的接种酵母。在28℃下，旋转（180 rpm）的孵育过夜。测量在过夜培养物在600nm（OD 600）的光密度。稀释的酵母细胞至0.2在50毫升新鲜的选择性培养基的OD 600。继续在28℃孵育细胞，旋转，以获得1×10 9酵母细胞7。 </li><li> 利什曼原虫鞭毛，鲍在5mL的AM介质culate寄生虫用7.5％FCS（胎牛血清）。在24℃，旋转（180 rpm）的孵育过夜。测量过夜培养的OD 600。稀释寄生虫细胞至0.2在50毫升新鲜的选择性培养基的OD 600。继续在24℃下温育细胞，旋转，以获得5×10 8细胞寄生虫12。 </li><li>对于布氏锥虫 ，寄生虫接种在5mL SDM-79培养基含10％FCS和30μg/ mL潮霉素。在27℃下，旋转（180 rpm）的孵育过夜。测量过夜培养的OD 600。稀释寄生虫细胞在50毫升新鲜的选择性培养基的0.2 OD 600。继续在27℃下温育细胞，旋转，以获得5×10 8细胞寄生虫13。 </li><li>对于大肠杆菌 ，接种细菌在5ml含有100μg/ mL氨苄青霉素DYT培养基。在37℃下孵育过夜，旋转（180转）。测量过夜培养的OD 600。稀释的细菌细胞至0.2在50毫升新鲜的选择性培养基的OD 600。继续在37℃下孵育细胞，旋转，以获得5×10 10个细菌细胞14。 注:过夜孵育后，将细胞在培养可在任一对数或稳定生长期。非常缓慢生长的细胞株可能需要比一晚，或细胞的更大数量的接种更长的温育时间，如凭经验确定。 </li></ol></li><li>对于所有的细胞类型，在1500×g下将含有细胞的培养基转移到50ml聚丙烯管中离心3分钟。 </li><li>除去上清，重悬的所有细胞类型的沉淀在1ml的有关细胞培养基。 </li><li>离心机中在3900×g下1分钟的微量离心管中的所有细胞类型。彻底去除上清。 </li><li>置于冰上的细胞。 </li></ol>注:处理液氮小心。使用包括cryogloves和谷歌的个人防护装备。丙烷是潜在爆炸性，所以在一个通风良好的房间或下通风橱进行液化。禁止明火被允许。 <ol><li>把约150毫升的液体氮的在聚苯乙烯托盘中。放置一个黄铜杯（高度3.6厘米，直径为2厘米，厚1.5毫米）液氮冻结它。不要让液体氮渗入铜杯。 </li><li>等到泡沫消退，以确保黄铜杯被冻结。 </li><li>放置成与黄铜杯壁接触连接到所述气缸丙烷软管。 </li><li>轻轻打开丙烷气缸阀。 注:在此步骤中，液化丙烷在铜杯。 </li><li>通过关闭丙烷气​​瓶阀和快速除去气体软管停止液化。 </li><li>充满液体NI聚苯乙烯托盘从黄铜杯顶部可达5毫米。不要让液氮渗透到黄铜杯中。 </li></ol> 5.样品冻结<ol><li>在聚苯乙烯托盘中放入约200 mL液氮。将小黄铜杯（高1.5厘米，直径2.5厘米，厚度1毫米）放入液氮中。将一个杯子放入一个悬浮液样品中。小心不要混合样品。为此，将样品1放入杯子1，杯子2中的样品2 等 。 </li><li>通过将尖端浸入液氮中来冻结镊子。 </li><li>在3.5中获得的颗粒中插入双刃解剖针。 </li><li>使用镊子，取第一部分制备的形状为400目的铜网显微镜网格。 </li><li>使用解剖针，将一滴3.5中获得的颗粒放在网格上。尝试不同的液滴尺寸（ 例如 2,5和10μL）。 </li><li>将镊子保持的电网迅速浸入液态丙烷基因额定在部分4，并搅拌以圆形运动的网格为几秒钟。 </li><li>迅速用镊子转移冷冻格在5.1节冻结的小黄铜杯。 注:对于每个样品，进行至少3个格。采取了铜网之前，请务必冻结镊子。 </li></ol> 6.冷冻取代注:四氧化锇是潜在的易失性，所以使用空气净化呼吸器与蒸气墨盒。冷冻在跳水冷冻前液氮固定剂也是一种解决方案。 <ol><li>对于超微结构研究，打开包含在节2.1制备冷冻置换介质的1.8毫升离心管的管。用于免疫定位研究，打开包含在2.2节制备的冷冻置换介质的1.8毫升离心管的管。广场上的冷冻管帽。 </li><li>通过在液氮中切入其尖端冻结镊子。 </li><li>迅速取下瓶盖和转移冻结的网格我扎成使用镊子将冷冻管。 </li><li>把盖子重新拧上冷冻管。 </li><li>重复该操作对每个样品的每个网格。 </li><li>关闭所有的帽和搅拌冷冻管与所述手的一个圆周运动，以确保样品在冷冻置换介质。 </li><li>在冷冻 - 置换处理放置在冷冻管用于在-84℃冷冻机中3天气密箱。 </li></ol> 7.样品变暖<ol><li> 超微结构的研究 <ol><li>携带在聚苯乙烯托盘中的冷冻管（以避免温度的突然上升）到-30℃冷冻机中。将它们放置在-30℃冷冻1个小时。 </li><li>放置在-15℃的冰箱中的冷冻管中2小时。放置在4℃下将样品2个小时，然后在室温下30分钟。 </li><li>除去用塑料移液管冷冻置换介质。添加约500 100％丙酮的μL。 </li><li>搅拌1.8毫升冷冻小瓶与手的一个圆周运动，并迅速倒入含有在玻璃小瓶中的网格（高度为3厘米，直径为2厘米）的丙酮（1.5mL）中。 </li><li>冲洗用1mL 100％丙酮的相同冷冻小瓶，以确保已经传送的所有的网格。搅拌与手的圆形运动的冷冻小瓶，倒入离心管的内容到玻璃小瓶中。 </li><li>冲洗每个玻璃小瓶用3mL的100％丙酮3次，持续10分钟。 </li><li>按照嵌入和包容的操作程序，15说明。浸渍在丙酮中用增加环氧树脂的浓度（25％，50％和75％）的样品，并嵌入在明胶胶囊中100％环氧树脂的样品。 </li></ol></li><li> 免疫标记研究 <ol><li>携带在聚苯乙烯托盘中的1.8毫升离心管（以避免温度的突然上升）到-30℃冷冻机中。 </li><li>放置2小时的冷冻管在-30℃冷冻机中。 </li><li>在一个-30°<html> C冰箱，搅动冷冻小瓶与手的一个圆周运动，并迅速倒在聚丙烯小瓶（高度5厘米，直径的1.5厘米）的冷冻置换介质。 </li><li>用2毫升的新鲜冷冻置换介质的更换冷冻置换介质。 </li><li>放置聚丙烯小瓶在-30℃的冰箱中2小时。 </li><li>冲洗1个小时聚丙烯小瓶用2mL 100％丙酮中，并为用2mL的100％乙醇1个小时三次。 </li><li>按照嵌入和包容的操作程序，15说明。含浸丙烯酸树脂浓度的增加（25％，50％，在丙酮中75％）将样品和与嵌入在明胶胶囊中100％的丙烯酸树脂的样品。 </li></ol></li></ol> 8.可视化样本<ol><li>嵌入后，使样品为80nm的超薄切片用超薄切片。对比用2％的柠檬酸铅切片在室温下进行1分钟屁股= "外部参照"&gt; 15。 </li><li>使用80千伏或120千伏电子显微镜观察部分15。 </li></ol>

<ol>
	<li>Palade, G. E., Porter, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+endoplasmic+reticulum.+I.+Its+identification+in+cells+in+situ.">Studies on the endoplasmic reticulum. I. Its identification in cells in situ.</a> J Exp Med. 100 (6), 641-656 (1954).</li><li>Kellenberger, E., Johansen, R., Maeder, M., Bohrmann, B., Stauffer, E., Villiger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artefacts+and+morphological+changes+during+chemical+fixation.">Artefacts and morphological changes during chemical fixation.</a> J Microsc. 168 (Pt 2), 181-201 (1992).</li><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> Histochem Cell Biol. 130 (5), 877-889 (2008).</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=Out+with+the+old+and+in+with+the+new:+rapid+specimen+preparation+procedures+for+electron+microscopy+of+sectioned+biological+material.">Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material.</a> Protoplasma. 251, 429-448 (2014).</li><li>Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J Microsc. 203 (Pt 3), 285-294 (2001).</li><li>Bernales, S., McDonald, K. L., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+counterbalances+endoplasmic+reticulum+expansion+during+the+unfolded+protein+response.">Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response.</a> PLoS Biol. 4 (12), 2311-2324 (2006).</li><li>Kissov&#225;, I., Salin, B., Schaeffer, J., Bhatia, S., Manon, S., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+non-selective+autophagic+degradation+of+mitochondria+in+yeast.">Selective and non-selective autophagic degradation of mitochondria in yeast.</a> Autophagy. 3 (4), 329-336 (2007).</li><li>Gilkey, J. C., Staehling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+ultrarapid+freezing+for+the+preservation+of+cellular+ultrastructure.">Advances in ultrarapid freezing for the preservation of cellular ultrastructure.</a> Microsc Res Tech. 3 (2), 177-210 (1986).</li><li>Nogales, E., Scheres, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM:+A+Unique+Tool+for+the+Visualization+of+Macromolecular+Complexity.">Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity.</a> Mol Cell. 58 (4), 677-689 (2015).</li><li>Baba, 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+in+yeast.">Electron microscopy in yeast.</a> Methods Enzymol. 451, 133-149 (2008).</li><li>Leunissen, J. L. M., Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-pressurized+rapid+freezing+(SPRF):+a+novel+cryofixation+method+for+specimen+preparation+in+electron+microscopy.">Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy.</a> J Microsc. 235 (1), 25-35 (2009).</li><li>Lefebvre, M., 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=LdFlabarin+a+new+BAR+domain+membrane+protein+of+Leishmania+flagellum.">LdFlabarin a new BAR domain membrane protein of Leishmania flagellum.</a> PLoS One. 8 (9), 1-12 (2013).</li><li>Tetaud, E., 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=TbFlabarin,+a+flagellar+protein+of+Trypanosoma+brucei,+highlights+differences+between+Leishmania+and+Trypanosoma+flagellar-targeting+signals.">TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.</a> Exp Parasitol. 166, 97-107 (2016).</li><li>Wasmer, 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=Solid-state+NMR+spectroscopy+reveals+that+E+coli+inclusion+bodies+of+HET-s(218-289)+are+amyloids.">Solid-state NMR spectroscopy reveals that E coli inclusion bodies of HET-s(218-289) are amyloids.</a> Angew Chem Int Ed Engl. 48 (26), 4858-4860 (2009).</li><li>Lefebvre-Legendre, L., 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=Failure+to+assemble+the+alpha+3+beta+3+subcomplex+of+the+ATP+synthase+leads+to+accumulation+of+the+alpha+and+beta+subunits+within+inclusion+bodies+and+the+loss+of+mitochondrial+cristae+in+Saccharomyces+cerevisiae.">Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae.</a> J Biol Chem. 280 (18), 18386-18389 (2005).</li><li>Paumard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ATP+synthase+is+involved+in+generating+mitochondrial+cristae+morphology.">The ATP synthase is involved in generating mitochondrial cristae morphology.</a> EMBO J. 21 (3), 221-230 (2002).</li><li>Gabriel, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fox2-dependent+fatty+acid+&#223;-oxidation+pathway+coexists+both+in+peroxisomes+and+mitochondria+of+the+ascomycete+yeast+Candida+lusitaniae.">A Fox2-dependent fatty acid &#223;-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae.</a> PLoS One. 9 (12), 1-27 (2014).</li><li>Dubochet, 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+physics+of+rapid+cooling+and+its+implications+for+cryoimmobilization+of+cells.">The physics of rapid cooling and its implications for cryoimmobilization of cells.</a> Methods Cell Biol. 79, 7-21 (2007).</li><li>Meryman, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+living+cells:+principles+and+practice.">Cryopreservation of living cells: principles and practice.</a> Transfusion. 47 (5), 935-945 (2007).</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 Cell Biol. 88, 151-164 (2008).</li><li>Bobik, K., Dunlap, J. R., Burch-Smith, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+high-pressure+freezing+and+quick+freeze+substitution+of+plant+tissues+for+transmission+electron+microscopy.">Tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy.</a> J Vis Exp. (92), e51844 (2014).</li><li>Pinan-Lucarr&#233;, B., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+autophagy+in+the+filamentous+fungus+Podospora+anserina.">Monitoring autophagy in the filamentous fungus Podospora anserina.</a> Methods Enzymol. 451, 251-270 (2008).</li><li>Follet-Gueye, M. L., Pagny, S., Faye, L., Gomord, V., Driouich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+chemical+fixation+method+suitable+for+immunogold+localization+of+green+fluorescent+protein+in+the+Golgi+apparatus+of+tobacco+Bright+Yellow+(BY-2)+cells.">An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.</a> J Histochem Cytochem. 51 (7), 931-940 (2003).</li><li>Jesus, D. M., Moussatche, N., Condit, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+high+pressure+freezing+and+freeze+substitution+method+to+preserve+the+labile+vaccinia+virus+nucleocapsid.">An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.</a> J Struct Biol. 195 (1), 41-48 (2016).</li></ol>

作者宣称，他们没有利益冲突。

TEM是细胞器，细胞，和组织的超微结构观测的有效方法。冷冻固定/冷冻取代是目前两种超微结构和蛋白的抗原性的保存的最佳方法。化学固定剂渗透和行动非常缓慢，从而使结构重组的超微结构2的完全稳定之前。相反，冷冻固定/冷冻置换瞬间稳定蜂窝结构4。然而，冷冻固定/冷冻替代技术包括了许多限制和困难从而限制了它们的应用范围的。这涉及样品，冷?...

样品制备为任何电子显微镜研究成功的关键。常规EM固定是用于固定的组织或细胞进行透射电子显微镜（TEM）1的主要方法。首先，醛和四氧化锇用于化学固定在室温下该材料。然后，将材料脱水用有机溶剂，渗透，并包埋在环氧树脂中。此方法取决于固定剂的渗透速率进入细胞。因此，细胞内容物中的工件和提取通常被观察到2个。 冷冻固定显然是细胞结构3保存一个更好的选择，让他们完好。可以使用冷冻固定/冷冻置换方法来获得在薄树脂切片TEM图像4的最高质量。这种技术的目的是为了获得玻璃化的双ological样品没有冰晶形成或含有冰晶小到足以不损伤细胞的超微结构。高压冷冻（HPF）和超快速冷冻固定，其也被称为切入冷冻（PF），两种方法来cryofix样品。 HPF在单元固定不动的分子瞬时且避免了由常规EM固定的损害。几种类型的冷冻机和自动替换设备已经开发5。冷冻机器和消耗品（液氮，标本的载体，等等）是昂贵的，但它们允许生产高品质的电子显微照片6,7。 PF是已在50年代初采用和在文献中简单和便宜8 .During的PF程序，将制备的样品以很快的速度冷冻，得到冰晶尺寸小于3至5nm，描述了一种技术。为此，所述样品是陷入液态制冷剂，如乙烷，丙烷，或乙烷 - 丙烷混合物。自50年代以来，PF的改进已经完成，以使这项技术提供给用户更大的数字。高压冷冻目前冻结大量的各种样品的厚度大于50微米（最多为盘形样品的厚度为200微米）5的唯一可行的办法，而PF被广泛地用于图像的小物体（&lt;100nm的），如悬浮于无定形冰9的薄膜大分子复合物。较大的样品，例如真核细胞，可以通过PF cryofixed，但需要试样保持器，如毛细管铜管或三明治系统8，10，11。 这里，快速，易于使用和可用于各种悬浮细胞培养物的低成本暴跌冷冻/冻干替代技术被呈现。

<table><tbody><tr><td>Grids</td><td>Electron Microscopy Sciences</td><td>T400-Cu</td><td></td></tr><tr><td>Formvar</td><td>Electron Microscopy Sciences</td><td>15800</td><td></td></tr><tr><td>Propane N35</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Double edge dissecting needle</td><td>Electron Microscopy Sciences</td><td>72947</td><td></td></tr><tr><td>Cryovials</td><td>Electron Microscopy Sciences</td><td>61802-02</td><td></td></tr><tr><td>Osmium tetroxide</td><td>Electron Microscopy Sciences</td><td>19130</td><td></td></tr><tr><td>Glass vial 8.5 mL</td><td>Electron Microscopy Sciences</td><td>64252</td><td></td></tr><tr><td>Uranyl acetate</td><td>Electron Microscopy Sciences</td><td>48851</td><td></td></tr><tr><td>Acetone</td><td>Sigma</td><td>32201</td><td></td></tr><tr><td>Ethanol 100%</td><td>Sigma</td><td>32221</td><td></td></tr><tr><td>Glass slides</td><td>VWR international</td><td>631-9439</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Acrylic resin</td><td>Electron Microscopy Sciences</td><td>104371</td><td></td></tr><tr><td>Epoxy resin M</td><td>Sigma</td><td>10951</td><td></td></tr><tr><td>Epoxy resin M hardener</td><td>Sigma</td><td>10953</td><td></td></tr><tr><td>Dibutyl phtalate&amp;nbsp;</td><td>Sigma</td><td>80102</td><td></td></tr><tr><td>Epoxy resin M accelerateur</td><td>Sigma</td><td>10952</td><td></td></tr><tr><td>Crystallizer</td><td>Fischer scientific</td><td>08-762-9</td><td></td></tr><tr><td>Joseph paper</td><td>VWR international</td><td>111-5009</td><td></td></tr><tr><td>Brass cups</td><td></td><td></td><td>do it yourself shop or made by yourself</td></tr><tr><td>&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Shizosacharomyces cerevisiae&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Trypanosoma brucei&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Leishmania amazonensis&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&amp;nbsp;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Airtight box</td><td>Fischer scientific</td><td>7135-0001&amp;nbsp;</td><td></td></tr><tr><td>Air purifying respirator</td><td>Fischer scientific</td><td>3M 7502&amp;nbsp;</td><td></td></tr><tr><td>Cartridge for respirator</td><td>Fischer scientific</td><td>3M 6001</td><td></td></tr><tr><td>Particulate filter</td><td>Fischer scientific</td><td>3M 5N11&amp;nbsp;</td><td></td></tr></tbody></table>

plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

透射电子显微镜（TEM）是研究细胞超微结构，以本地化的蛋白质和以非常高的分辨率可视化大分子复合物不平凡的工具。但是，要获得尽可能接近原生状态，需要完善的样品保存。与醛常规电子显微镜（EM）固定，比如，不能提供良好的超微结构保存。固定剂的缓慢渗透诱导细胞重组和各种细胞成分的损失。因此，常规EM固定不允许结构和抗原性的瞬时稳定和保存。审查细胞内事件的最佳选择是使用冷冻固定，然后，保持细胞的天然状态的冷冻替代固定方法。高压冷冻/冻干取代，其保留细胞的超微结构的完整性，是最常用的方法，但需要expensi已经装备。这里，易于使用和低成本的冷冻固定方法进行冷冻取代悬浮细胞培养物呈现。

在本文中，提出了一种用于超微结构（ 图1和图2）和免疫标记（ 图3）的研究一种易于使用和低成本的暴跌冷冻方法。我们证明，这是没有必要有专用设备冷冻置换程序，该程序升温只需不到6个小时，而不是使用专用系统的24个小时。 PF /冷冻置换方法导致在<...

Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

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

此手稿描述了通过透射电子显微镜可视化用混悬剂细胞中易于使用和低成本的冷冻固定方法。

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 ...

plunge-freezing-tool-for-ultrastructural-immunolocalization-studies

Research

JoVE Journal

Biology

10.8K 次观看.  Université de Bordeaux. 此手稿描述了通过透射电子显微镜可视化用混悬剂细胞中易于使用和低成本的冷冻固定方法。 

视频: Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in 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

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

用于可视化神经突在冷冻水合状态下使用低温电子断层扫描准备初级神经元

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

神经科学

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

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

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 ...

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

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

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, ...

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 ...

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these techniques, cryo soft X-ray tomography (SXT) allows imaging whole cryo-preserved cells in the water window X-ray energy range (284-543 eV), in which carbon structures have intrinsically higher absorption than water, allowing the 3D reconstruction of the linear absorption coefficient of the material contained in each voxel. Quantitative structural information at the level of whole cells up to 10 &#181;m thick is then achievable this way, with high throughput and spatial resolution down to 25-30 nm half-pitch. Cryo-SXT has proven itself relevant to current biomedical research, providing 3D information on cellular infection processes (virus, bacteria, or parasites), morphological changes due to diseases (such as recessive genetic diseases) and helping us understand drug action at the cellular level, or locating specific structures in the 3D cellular environment. In addition, by taking advantage of the tunable wavelength at synchrotron facilities, spectro-microscopy or its 3D counterpart, spectro-tomography, can also be used to image and quantify specific elements in the cell, such as calcium in biomineralization processes. Cryo-SXT provides complementary information to other biological imaging techniques such as electron microscopy, X-ray fluorescence or visible light fluorescence, and is generally used as a partner method for 2D or 3D correlative imaging at cryogenic conditions in order to link function, location, and morphology.

Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented.

通过冷冻软X射线断层扫描对细胞进行3D制图描述

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.