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>

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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. 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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，冷冻伪像，实现了技术，成本和设备。高压冷冻/冻干替代技术已使高度的蜂窝细节6保存，7。相比之下，PF已经用于OBS欧文悬浮于无定形冰9的薄膜小物体。这里，用于超微结构和免疫标记研究PF /冷冻替代方法提供了酵母，寄生虫和细菌超微结构媲美高压冷冻图像的高质量的图像。 固定质量取决于冷却速度18。在高的冷却速率，水是在玻璃态，并在电子显微镜水平没有显示出可见的晶体结构。玻璃化以非常薄的表面层（200μm的总称）单独出现，因此，良好的厚度冷冻会逐渐从样品到内层4的表面减小。在植物细胞中，填充有气体蜡质包衣或空间可以减缓冷却速度。与PF，令人惊讶地，观察到的冷冻性能的质量没有降低。在所有的实验，以及冷冻细胞的百分比为g<html>超过50％reater。这个百分比50％之间变化，以超过90％，并且是依赖于各种参数（培养基，细胞的生理状态，室外温度和湿度水平）。如上所述，冰晶的形成破坏细胞结构和冰晶在液泡特别明显。在PF /冷冻置换过程中，特别要注意的冻结温度。冷冻置换步骤必须不高于-82℃下进行，否则，玻璃体水将被转化成结晶水。为了避免冰晶体损伤的形成，冷冻保护剂是目前使用的，但其导致改变的细胞19的超微结构。酵母，细菌和寄生虫培养基可能包含一个组件，它是低温保护剂。在这种情况下，它不是决定使用额外的冷冻保护剂，以将尽可能接近实际的细胞状态。 PF /冷冻替代的成功取决于该方法开发阶段的许多关键步骤，首先，关键点是获得适当的颗粒稠度，离心后获得的颗粒必须足够紧凑，以便滚动如果颗粒太紧，冷冻置换溶液不能渗透液滴，超微结构不会保持良好，相反，如果颗粒不够紧凑，剩余的培养基可能会发育为了获得来自不同细胞类型的丸粒的适当一致性，有必要遵守培养条件以获得指定数量的细胞和所有方案中所示的离心参数（见上文）但是，放在铜格栅上的材料量不是关键的，可以测试不同的液滴尺寸;二，格栅类型t被用作支持是重要的。几个金，镍，和铜网格具有不同网眼尺寸进行了测试。最好的结果是用薄条的400网目铜网上获得。第三，关于冷冻步骤，黄铜杯优选液化丙烷。铜杯液体丙烷趋于冻结，因此，实验复杂化。最后，操纵者必须迅速在寒冷条件下工作，以避免意外样品热身。如果它发生，样品必须被丢弃。例如，为了防止样品升温，镊子必须在使用前预冷却和冷冻置换介质必须在手术前遥遥领先制备。不结冰的镊子实现冷冻镊子还必须在跳水冷冻过程becauseif暴跌冷冻用，大量的烟雾形式的镊子在丙烷骤降（由于热/冷交换）。这不可避免地对冷却速度的影响。与SAMPL测试与解冻冷冻镊子ES似乎不正确冻结。 此外，使用危险化学品如O 2 S O 4和乙酸双氧铀中冷冻置换介质需要，溶液的制备过程中并冷冻置换工序，具有充足的PPE通风柜下工作。为了防止的OsO 4的蒸气的泄漏和吸入，冷冻管必须有一个硬的O形环，以确保管保持期间冷冻置换处理密封。为了防止冷冻置换介质的制备过程中不必要的暴露于锇蒸气，所述的OsO 4安瓿可以在玻璃烧瓶被打破。在夹杂物后的树脂块玻璃碎片的存在可以通过在丙酮漂洗和嵌入的步骤，使用细尖移液管来避免。进行双眼循环下列入步骤和实现半薄切片还允许用于验证不存在玻璃碎片。当实验不能通风橱（特别是在冷冻置换方法的第一工序）下进行，使用具有蒸汽筒的空气净化呼吸器。在用液氮或在冰冻的步骤寒冷条件下工作时，机器人也必须小心。推荐使用手套和眼镜。此外，丙烷是潜在的爆炸性，所以液化必须在通风良好的房间或下通风橱中进行并没有明火是允许的。 几篇文章表明，在多种样品脑组织，常春藤叶，或拟南芥和烟草本塞姆氏的根尖如，超微结构的保存与HPF技术改进相比化学固定20,21。与PF，该实验是在悬浮液中的细胞进行。因此，不存在上拉这项技术的成功的指示rger标本样组织。进一步的实验需要进行，以解决这一点。然而，该方法已经成功地与不分离的细胞，但相当的菌丝形式22的丝状真菌壳属委陵菜测试。 在这篇文章中介绍的方法代表了化学固定或HPF当前方法显著的改善。例如，PF并不需要昂贵的设备或耗材的对比HPF 23。护理还必须采取由于HPF技术使用大量的液态氮（每实验约80升）。与所提出的技术，一个主要优点是没有重要的设备是必要的23。因此，实验的成本要低得多（约一百便宜倍）。另一个优点是，不需要特殊专长，因为该技术易于使用。此外，样品处理Ò˚FPF /冷冻取代比HPF /冷冻置换短得多由于变暖过程，需要小于6小时而不是24小时24。这是不耗费更多的时间比传统的化学固定。的优点是，该细胞的超微结构是保存完好的具有最小伪影和更接近该单元的原来的状态。该技术的应用肯定会使用来研究不同的快速生物事件或以高分辨率本地化蛋白在悬浮液中生长的细胞和几微米的厚度，样品的诸如真菌菌丝22。

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酿酒酵母中（ 图1A和1D-H）， 粟酒裂殖酵母 （ 图1B和细胞内结构的改善的保存 C）， 利什曼原虫鞭毛 （ 图2A 和 B）， 布氏锥虫 （ 图2 C和D），和大肠杆菌 （ 图2E）。结构没有缩小，细胞质出现密集。此外，该膜是光滑的，这是很好的保存证据。所有的细胞器，如细胞核（ 图1A，B D和图2A-D），线粒体（ 图1A-C，E和H），和液泡（ 图1A，C，F和H）的，可完美识别。在细胞核中，双膜形成核膜，核孔，纺锤体极体，和安装在核膜的外膜核糖体清晰可见（ 图1D和图2B，2D）。在线粒体中，内部膜内陷，称为嵴（ 图1E），都清晰可见。液泡是细胞器至关重要，因为大冰晶可以在里面他们很容易地开发。 如图1所示，液泡保存完好无冰晶体损伤（ 图1A，C，F，和H）。最后，如超微结构保存完好，不同的生物事件可以被观察和分析，如自体吞噬（ 图1C，F和H），线粒体形态（ 图1E），分泌囊泡（ 图1G），以及细胞核分裂（ 图1A 图2 B，一个ND 2C）。 在用特异性抗体蛋白的原位定位是在细胞生物学中最强大的和流行的技术之一。 PF也可以用来作为一种方法来执行蛋白免疫定位。此过程保持蛋白的抗原性，正如上文提到的，细胞器的身份。不同的抗体针对不同的酵母蛋白，如抗ATP合酶7，15（ 图3A，3C和3D），抗GFP（ 图3B），抗porine 16（ 图3 E），和抗FOX2 17（ 图3F），已经过测试。另外，也可以在不同的生物体，如利什曼原虫12执行这些实验。 图1: 酵母 酿酒 酵母 和 裂殖 酵母 的超微结构研究的TEM照片 。 （A） 酿酒酵母的概述。 粟酒裂殖酵母的（B）概述。 粟酒裂殖酵母细胞质的（C）部分。 （D）一种核的高倍放大。 （E）详细嵴清晰可见线粒体的。自噬过程中（F）液泡形态。 （G）酵母花蕾内侧分泌囊泡。在自体吞噬过程液泡和线粒体之间的紧密接触的（H）。实施例。 N，核; V，液泡;米，线粒体; SV，分泌VESI克莱斯; R，核糖体。比例尺= 200纳米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54874/54874fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/54874/54874fig2.jpg" /> 图2: 利什曼原虫amazonesis， 布氏锥虫 和 大肠杆菌 的超微结构研究的TEM图像 。 （A） 的利什曼原虫的概述。 利什曼原虫细胞质的（B）详细地核孔和内质网。 布氏锥虫的（C）概述。 （D） 布氏锥虫核的高倍放大。 大肠杆菌细胞的（E）概述。 N:细胞核; V，液泡;米，米托chondrion; NP，核孔;男，鞭毛; FP，鞭毛袋; K，动基体; R，核糖体。比例尺= 200纳米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54874/54874fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/54874/54874fig3.jpg" /> 图3: 使用单克隆和多克隆抗体的蛋白质标记的实例。 （A） 酿酒酵母的概述。淀粉样蛋白β的（B）免疫标记与抗GFP抗体。 （CD）用抗ATP合酶抗体线粒体标记的实施例。 （E）孔蛋白定位在线粒体的外膜。 （F）FOX2蛋白在线粒体通过免疫标记检测。 Ld的 Flabarin的（G）中的本地化利什曼原虫amazonesis的鞭毛的纵截面。所有抗体都在使用10nm的金多克隆抗体检测到。 N，核; V，液泡;米，线粒体。比例尺= 200纳米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54874/54874fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Universidad San Sebastian

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

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 ...

科研

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 ...

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 ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

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 ...

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, ...

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 ...

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.