Name: fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy
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This presentation was supported by a Clinical Innovator Award to JLC from the Flight Attendant Medical Research Institute and by the United States Environmental Protection Agency. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Cooperative Agreement CR83346301 with the Center for Environmental Medicine, Asthma, and Lung Biology at The University of North Carolina at Chapel Hill, it has not been subjected to the Agency&#8217;s required peer and policy review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

1，制备生物样品的冷冻断裂/冷冻蚀刻<ol><li>使用常规EM固定剂配方如2％戊二醛+ 2％多聚甲醛的0.1M磷酸盐缓冲液1小时。进行生物体组织的主要固定。注意:虽然它可能是可取的，冻结某些类型的样品无需事先固定的，普遍血液传播病原体的预防措施强制适当的固着，其中试样由人体组织。 </li><li>以下主要定影，冲洗样品在补充有0.2M的蔗糖不超过1小时，在相同的缓冲液。 </li><li>样品转移到在0.1M磷酸盐缓冲液组成的25％甘油的冷冻保护剂溶液为不超过1小时以上。 </li></ol> 2，冷冻和标本的存放在冷冻断裂的研究进展<ol><li>的适当大小和形状的试样选择金或铜试样存根被处理（ 图4中 </st荣&gt;）。 </li><li>使用细级镊子和/或在金属试样存根顶部的试样的小轨距的注射器针头位置的小片段。 <ol><li>减少样品的液体含量为粘性的，略微粘状稠度通过从用滤纸短截线的边缘处撤出液体。 </li><li>对于单副本，用小规格的注射器针头上的存根创建组织的土丘。对于双副本，反转另一桩和位置也正好在存根，轻轻放置到样品表面创建一个三明治（ 图5A）。不要按样品从两个桩之间。 </li></ol></li><li>填写两根绝缘的杜瓦瓶液氮。注:第一艘可容纳含小灵通是用来从市售缸（ 图5B）液化少量丙烷气的金属柱。第二个容器是一个分区的存储/保持器的简要LY保持在液氮下冷冻样品。 <ol><li>使用定位在丙烷以及气缸喷嘴，打开气瓶阀和使气体流入所述第一容器中，其中，将液化的阱。注意:小心！丙烷爆炸，可能会造成冻害开接触，是一种窒息。仅在化学罩用于这种用途照顾也避免了外来火源，用衣服保护防寒，保持足够的通风认证使用丙烷。 </li><li>尽可能快地，拿起试样安装与细级镊子和沉浸入液化气体的第一容器几秒钟，然后很快转移到液氮中的第二保持器（ 图5B）。 </li><li>一旦样品被冻结，液态氮，直到准备下存储转移到冷冻断裂装置。存根转移到一个更大的存储液氮杜瓦容器扩展如果需要的话，但是关心存放时，应注意不要让存根解冻。 </li></ol></li></ol>冷冻断裂3。操作仪器和样品处理<ol><li>在黄铜保持器并进入腔室中的标本<ol><li>加载金试样存根成小册子型双复制品试样保持器或液氮下夹紧装置和保持在那里，直到准备在试样腔室（ 图6）来定位。 </li><li>当腔室已达到高真空（约2×10 -6毫巴）及阶段已被冷却至液氮温度，则关闭泵，排气腔室，并尽可能快地定位安装试样存根到样品表。 </li><li>启动泵，重新建立在高真空，并使用电子级和手臂温度控制来调节样品台温度为-100℃，并直接液氮到切片机臂和BRI它纳克至液氮温度。 </li></ol></li><li>的断裂过程 <ol><li>当在液氮温度下实现高的真空度，-100ºC阶段的温度和切片机臂，远程打开双复制品试样保持器，从而压裂在试样架的标本（冷冻fracture_movie1.mov）。 </li><li>在标本用于蚀刻下骨折的情况下，使用保持在夹紧刀片在切片机臂刮（ 即骨折）的标本，以代替步骤3.2.1表面（冷冻etch_movie2.mov）。 </li><li>如果添加的冷冻蚀刻步骤将被执行，放置冷却，切片机臂在断裂面为一至数分钟。注意:此动作会升华（ 即蚀刻）水从断裂面。这个动作的效果是揭示真实细胞表面，细胞外基质，和/或分子组装的水升华，除了FRActure面孔穿过细胞膜的脂双层。 </li></ol></li><li>金属遮蔽和副本支持蒸发用电子枪 <ol><li>激活的铂/碳的电子或电阻枪，并允许它从30度的角度蒸发一薄层铂/碳（约2纳米）以上的断裂面 - 45°。这通常需要大约15 - 20秒。 </li><li>激活的碳电子或电阻枪如步骤3.3.1和蒸发一层薄薄的碳的断裂试样表面的直接开销（90度）给予副本的支持。这通常需要大约15 - 20秒。 </li></ol></li><li>副本检索<ol><li>当复制阴影和碳稳定后，关闭真空泵，发泄室，取出试样从仪器安装。 </li><li>使用一对细粒级镊子从双删除的每个黄金标本存根副本小册子/夹，并允许前，轻轻地降低存根到水面上的斑点板（ 图7）解冻几秒钟。注:碳/铂副本包含附着的样品材料会浮到水面。 </li><li>操纵使用一个细金属丝环，或通过细级镊子和转移到另一个点板含5％重铬酸钠在50％硫酸为一至数小时保持以往的铜电子显微镜网格中的副本。注:此浴从副本消化的实际生物样品材料。充分强度的家用漂白剂可被取代为重铬酸盐/硫酸溶液。 </li><li>当消化完成后，传送回副本到一个干净的水面，检索到一个标准的铜电子显微镜网格。 （注:副本可​​能会碎裂成小块，在消化过程中，但即使是非常小的副本可能包含大量的信息，并应RETRieved和检查。）店铺副本在市售格箱，他们将保持稳定多年，柔和处理配套电网。 </li></ol></li></ol>冷冻断裂/蚀刻副本4超微结构检查<ol><li> 80千伏 - 视图中的透射电子显微镜在加速电压通常为50复制品。 </li><li>记录在标准的TEM薄膜或用高分辨率的数码相机相关的图片。 </li></ol>

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The author has nothing to disclose.

在下面的介绍和商业可用性的岁月，冷冻断裂/蚀刻方法被广泛用于生物膜结构的调查。事实上，一些膜的结构特化的最佳角度已在冷冻断裂/蚀刻制剂获得。这些研究不仅有助于细胞膜的结构组织的了解也深入介绍了如何结构和功能有关。 常规生物电子显微镜的出现所带来的认识，与醛固定剂，反应性化学物质，并赋予差分电子密度的生物标本所需的重金属的化学处理本身对伪影的产生作出贡献。的冷冻断裂技术的发展是部分基于努力减少加工工件。然而，已经认识到，冷冻断裂/蚀刻推出了自己的一套文物，其中一个主要的一个是冰晶破坏。组织直接置于液态氮经历减小的冷却速度由于氮气的周围这导致抹杀超微结构细节冰晶的形成样品的绝缘层的形成。这个问题是克服与使用冷冻保护剂如甘油和通过在液氮中冷却的丙烷第一冷冻组织。从历史上看，在液氮中进行切入冷冻冷却氯氟烃但这些材料已被淘汰使用的，因为它们对环境的不良影响。即使在最好的冷冻保护的条件下，该方法的性质不可避免地导致粗大，砾石等表面胞质骨折;但是，通过膜结构裂缝揭示具有特定平面的组织相对于它们在细胞膜上取向良好的组织特征。这些图像是可能只具有使用冷冻保护剂如甘油，限制冰晶体的损害，和甘油本身在某些情况下也可能会引入相关联的隔膜颗粒的人为的再分配。此外，甘油不能从断裂面从而限制了机会成功蚀刻升华。如果在随后的蚀刻过程是可取的，则该试样必须是在一种时尚，使用任一液态氦或氮搪塑限制冰晶体损伤快速冷冻和/或接近所使用的冷冻保护剂，如丙酮被容易地从破碎的升华表面。 虽然转向的初步报告都集中在成像的病毒，它是在膜结构的冷冻断裂/蚀刻找到更广泛的应用。事实上，结构，如紧凑，缝隙连接和纤毛膜专业化已经显露典雅，在冷冻断裂的准备。冷冻断裂有founð相当的实用程序在开发过程中15，17,24和膜病理25-30取得膜分化的理解。从这个实验室以前的报告中，已经证明了紧密的连接复合体差异化气道发育过程中的模式，与实验感染因子暴露有关纤毛膜结构，以及在呼吸道上皮细胞紧密的连接复合体与空气污染和接触烟草烟雾有关的降解破坏在实验动物和人体实验对象。 该技术扩展冷冻断裂/冷冻蚀刻过程 使用液化丙烷冻结此处描述的标本的淬火方法也许是最常用的方法，以冷冻生物样品的常规冷冻断裂。然而，其他的方法来冷冻必须拨其中冷冻蚀刻将被执行主编。因为甘油是一种非etchablecryoprotectant，标本为冷冻蚀刻必须的方式，限制了冰晶形成被冻结。这可以通过使用etchablecryoprotectant如丙酮和/或冻结的样品座对金属表面的冷却用液态氦或液态氮搪塑来完成。无冷冻保护剂快速冷冻也可使用喷雾冷冻或高压液化丙烷冷冻来完成。 最近进一步与结构和功能使用冷冻断裂技术的努力已拨付这种技术协同免疫组化标记31,32。这里，抗体对特定抗原可有效地，准确地定位于膜位点（ 图11）。 虽然冷冻断裂与单向阴影是目前使用最广泛的协议，一些实验性的设计将要求迅速冻结了little或有限的抗冻剂，伴随着蚀刻的断裂面露出真细胞表面，细胞外基质，和/或大分子复合物，和旋转遮蔽赋予对比度16（ 图12和图13）。 有一个在冷冻断裂的纳米技术，材料科学的应用越来越感兴趣，并在使用这种技术在分子生物学的成像工具。事实上，在许多方面的纳米材料可能会在他们的组织与该冻结破裂的最初设想作为成像手段病毒的结构是一致的。此外，制造的脂质体，在形式与生物膜相似的结构，现正被广泛研究用于靶向递送的药物试剂和定向基因治疗。因为他们的组织基本上是细胞膜一样，冷冻断裂的处理可能在生产揭示图像作为开发板的一个组成部分相当实用ping通这些疗法33。 综上所述，冷冻断裂技术已在表征细胞膜的组织和功能是重要的历史意义。然而，冷冻断裂是找到与分子生物学和纳米技术，几乎可以肯定的协调工具的新领域将在这些迅速崛起的领域做出贡献的进步。

这个概念和生物标本冷冻断裂处理实际应用一个世纪前引入了斯蒂尔1半以上。早期的设备，不同的拨款组件集成到一个工作独立单元1。原装置进行了修改，并精制成商业上可用的工具，以容纳用于远程操作，维修高真空的迫切需要，并且碳和金属的蒸发，以产生由透射电子显微镜（ 图1和图适合于考试的复制品2）。 典型的仪器由一个高真空室与样品表和（ 图1）切片机臂具有可调节的液氮通过量。该腔室还容纳两个电子枪，一个用于稳定碳蒸发定位在90度角到试样阶段，另一个用于普拉蒂NUM /碳遮蔽在一个可调节的角度，典型地15° - 45°（ 图2）。本机的电源被应用到操作真空泵及电子面板调节温度调节和电子枪控制。 最初的设想为手段，以实现病毒改进成像，冷冻断裂甚至获得了更多的人气，作为参加考试和细胞膜的分析和自己的专业2,3的技术。事实上，此过程一直积分来阐明细胞和组织中，许多这些研究站，以细胞和分子生物学4-9经典捐款的结构/功能关系。用于冷冻断裂技术的发展的主要目标和理由是限制工件可观察到的，在电子显微镜的分辨率从化学固定和加工中常规生物电镜用于导出。这里的目标是限制化学固定和冷冻以足够的速度和频繁的冷冻保护剂，以限制冰晶形成和其它冷冻的工件存在检体。最近，这一技术已经发现了从分子生物学家和材料科学的调查审核纳米粒子和纳米材料的兴趣死灰复燃。 冷冻断裂和冷冻蚀刻图像呈现出立体字，有时被误认为是扫描电子显微照片。然而，冷冻断裂制剂通过透射电子显微镜观察和高分辨率的形态学研究的主要贡献是细胞膜的结构/功能元件的独特表示。冷冻断裂处理是通过冷冻的细胞和组织具有足够的速度以限制冰结晶和/或与使用冷冻保护剂如甘油引发。试样，然后在真空和代理商断裂LICA是由碳和铂在断裂面的蒸发产生的。最初的样本是从被检索到一个标准的EM试件格子副本消化。冷冻断裂图像的另一种常见的误解是，它们显示了细胞表面上。然而冷冻断裂的基本前提是，生物膜穿过脂双层的破裂过程（ 图3）分离。这个过程在生物膜产生2断裂面，其中一个显示邻近于细胞质中，PF-面在膜的一半的组织，并且其中一个显示在膜的双分子小叶的一半，相邻的外氛围中，EF面。真细胞表面没有在冷冻断裂图像表示，但只有当冷冻蚀刻后的断裂过程的后续步骤中加入用于显示。为了有效地刻蚀先前断裂试样以显示表面德泰L，样品必须以极快的速度，没有unetchablecryoprotectant被冻结。从断裂试样揭示标的特征的表面蚀刻的水被定位在冷却切片机臂在样品阶段创建阶段之间的温度差保持在检体和所述冷却切片机臂，导致​​水从表面升华来实现的。当水从断裂试样的表面上的冷冻蚀刻机动过程中升华，然后实际细胞表面，细胞外基质，细胞骨架结构和分子组装的各方面可以以高分辨率显示。因而冷冻断裂和冷冻刻蚀不能互换术语而是反映逐步的过程，其中后者可以不依赖于特定研究的需要是必须的或可取的。 以下的冷冻断裂/冷冻蚀刻程序，断裂表面进行定向evapora略去碳和铂的外套，以提供支撑和成像对比的复制品。铂/碳成像蒸发可以是单向的或旋转​​，并通过任一电阻或电子枪来实现的。从已知的角度，一般30度单向阴影 - 45度，是在执行某些形态的计算是有用的。已进行深蚀刻试样通常是旋转遮挡和这些样品的所得图像评价是照相反转。 的历史以及在冷冻断裂/冷冻蚀刻技术的当前目标是限制化学固定和处理样品是与更常规的透射式电子显微镜的程序相关联的伪影。然而，这种技术提供了在其赋予三维细节，从而促进采集生物，材料科学形态的数据，并且n的能力的实质性优点anotechnology标本。冷冻断裂和冷冻蚀刻过程是复杂的，多方面的，其应用的某些方面进行定制。此演示文稿提供了进程的主要特点的调查来看，读者被称为全面的出版协议10，11，以解决细节和定制过程中对特定的研究需求。

<table><tbody><tr><td>Balzers Freeze-fracture/freeze-etch plant</td><td>Balzers</td><td>BAF400T</td><td></td></tr><tr><td>Standard buffers</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Standard aldehyde fixatives</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Sodium dichromate</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Sulfuric acid</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Disposable supplies for Platinum/Carbon Evaporation</td><td>Technotrade International</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Propane</td><td>various suppliers</td><td></td><td></td></tr><tr><td>Disposable supplies for electron microscopy</td><td>Electron Microscopy Sciences</td><td></td><td></td></tr><tr><td>Transmission electron microscope</td><td>Carl Zeiss Inc.</td><td></td><td></td></tr></tbody></table>

fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy

冷冻断裂/冷冻刻蚀描述由此标本，通常在自然界中的生物或纳米材料，冷冻，破碎，和复制，以产生碳/铂"铸造"用于检查通过透射电子显微镜的方法。标本进行超速冷冻速率，往往在冷冻保护剂的存在下，以限制冰晶形成，与样品的后续压裂在液氮冷却的高真空条件下的温度。所得到的断裂面进行复制，并从该表面赋予立体细节来铸造一个角度稳定的由碳和铂的蒸发。这种技术已被证明为细胞膜和其专业的调查特别有启发性，并大大的细胞形态相关细胞功能的理解做出了贡献。在这份报告中，我们调查了仪器的要求和技术协议执行冷冻断裂时，相关的命名法和断裂面的特征，变型的常规方法，和标准的冷冻断裂解读图像。这种技术已被广泛用于细胞生物学的许多领域超微结构的调查和有希望的分子，纳米技术的新兴成像技术和材料科学的研究。

冷冻断裂图像判读的关键前提是，断裂面穿过膜赋予2断裂面的脂质双层，称为按约定的PF-面（等离子断裂面）和EF-面（外断裂面）（ 图3）。的PF-面是相邻的小区和EF-面的细胞质中的膜脂双层的一半是邻近于细胞外环境的膜脂双层的一半。该冷冻断裂技术是膜结构的调查是特别有用的，并在两个表面都典型地与PF-面孔被填充在对比EF-面含有更少的许多膜相关联的颗粒明显不同。冷冻断裂细胞的胞质内容物在外观上通常粗的和没有特别揭示虽然膜结合的细胞器如细胞核和高尔基体可以容易地识别。特别感兴趣的INVE使用这种技术stigators是膜结构的特化，可以被解释为它们的功能。这些措施包括相关膜颗粒的具体分布，纤毛膜专业化和细胞间连接处。 纤毛结构相关 居住在细胞膜在每个真核纤毛和鞭毛的基部是相关联的隔膜颗粒的阵列组织成股线包围纤毛的轴和被称为纤毛项链（ 图8）12-16。该结构表现出某种程度的不同物种的形态学不均匀性和据推测，因为它出现之前，睫状轴ciliogenesis中的出现和保持在适当位置中的成熟纤毛是多官能因为。初生纤毛项链膜粒子阵列的聚集和组织之前，e的时间推导纤毛轴ciliogenesis提示其在发展轴丝14的早期组织的参与过程中融合。 紧密连接的上皮Permeabi lity 在各种上皮细胞，冷冻断裂研究表明吻合链和凹槽结构包围基底外侧边框的顶端方面的复杂的组织。这种带状结构被称为紧密连接或zonulaoccludens（ 图9），被认为是作为上皮通透性的细胞旁焊剂3，17-20的调节器。紧密连接了几股，而那些更复杂的组织被描述为"从紧"被形容为"漏"。该组织似乎适合于它们相关的上皮细胞的功能。该PF-面紧密连接显示为股而EF-面孔表现出complementary凹槽。 间隙连接和细胞间通讯 间隙连接（ 图10）出现在多种组织中冷冻断裂制剂，并通过对PF-面和互补的凹坑上EF-面颗粒的密集阵列来表示。这些结构被认为反映了膜位点，以促进细胞间通讯。在组织中，其中它们的存在，低分子量荧光染料和钙通量的通道的示范已证明21-24表明它们代表一个物理路由的信号传导分子的通过。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig1highres.jpg" width="500" /> 图1:布局商用冷冻断裂/蚀刻厂。 在最左边的是加压杜瓦˚Feeding冷却液氮的样品载物台和在受控条件下冷却切片机臂。在中心单元与试样腔室和电子控制面板的右侧。在右边是在其上的排放气被用于使样品腔室与大气相通的额外的液氮罐中。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig2highres.jpg" width="500" /> 图2:冷冻断裂/蚀刻植物标本室和门打开，露出定向瞄准电子枪冷却导流罩的位置，和标本的地方张贴标本，黄铜支架定位。样品通过端口引入室的左侧该腔室。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig3highres.jpg" width="500" /> <st荣&gt;的冷冻断裂的原理图3示意图。的断裂面，从该膜的通过脂质双层产生的细胞外表面（EF-面）和一个面，即最接近于细胞质的方面，分束派生PF面。真非裂缝膜表面仅通过蚀刻之后的冷冻断裂机动看出。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig4highres.jpg" width="500" /> 图4两种试样架的冷冻断裂/蚀刻过程。（左）双副本试样夹持小册子用于未蚀刻进行常规的冷冻断裂的过程。试样夹在两个金试样短截线之间并位于所述黄铜小册子。试样是通过在试样腔室真空下打开小册子压裂（参见冷冻FRACT<html> ure_movie1.mov）。 （右）的安装主要用于冷冻蚀刻。试样被定位在黄金存根并锁入黄铜保持架。试样是通过温和剃须用刀片固定在切片机臂断裂。该结构是优选的蚀刻程序（参见冷冻etch_movie2.mov）。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig5highres.jpg" width="500" /> 安装图5制备冷冻断裂试样，试样夹在两个黄金坐骑（A）之间，并冻结在一个良好含有液态氮冷却的丙烷（前景），随后转移至固定装有液氮的杜瓦（背景）（ B）。 ig6highres.jpg"WIDTH ="500"/&gt; 图6示范冷冻冷冻断裂试样定位到双复制品试样保持小册子在制备用于引入到冷冻断裂/蚀刻装置的样品室。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig7highres.jpg" width="500" /> 图7检索以下断裂和阴影中的冷冻断裂/蚀刻厂副本，"A"是一个干净的水面在现场板。从检体存根浮起的复制品被转移到"B"的良好含有重铬酸钠或全强度的家用漂白剂为从副本的组织的消化酸化溶液。 "C"表示后续干净的水表面，使清洁的副本之前转移到being检索到铜电子显微镜网格。 <img alt="图8" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig8highres.jpg" width="500" /> 图8纤毛通过冷冻断裂透露有关的结构。白色箭头指向上的上皮细胞发生ciliogenesis的管腔边界专门膜颗粒阵列。这些粒子阵列代表驻留在新兴和成熟的纤毛（黑色箭头）基地新生的纤毛项链。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig8highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig9highres.jpg" width="500" /> 图9断裂通过上皮细胞膜揭示<html>荷兰国际集团PF-和EF-裂缝面的例子。紧密连接复合体的链和凹槽组织是显而易见的，细胞间隙被标记的箭头。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig9highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图10" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig10highres.jpg" width="500" /> 图10对大鼠肝脏间隙连接的冷冻断裂形象。颗粒是显而易见的PF面和互补的坑都在EF面可见一斑。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig10highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 4 / 51694fig11highres.jpg"WIDTH ="500"/&gt; 用胶体金连接蛋白的图11免疫组化定位的冷冻断裂编制说明的缝隙连接特定的定位标记的抗体。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig11highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图12" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig12highres.jpg" width="500" /> 图12快速冷冻和深蚀刻和旋转遮蔽的一个例子。上皮细胞已通过细胞质被断裂和PF-面是显而易见的（箭头）。在后面的箭头可以看到一个真正的细胞表面通过后续的蚀刻透露。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig12highres.jpg" target="_blank">请CLI</a>完蛋这里查看该图的放大版本。 <img alt="图13" fo:content-width="5in" src="/files/ftp_upload/51694/51694fig13highres.jpg" width="500" /> 图13。快速冷冻深蚀刻和旋转遮蔽的例子，揭示包括牛气管ciliaryaxoneme分子复合物。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51694/51694fig13highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy at JoVE.com

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

基本的技术和用于检查通过透射电子显微镜生物标本和纳米材料的冷冻断裂加工的精炼进行说明。这种技术对于揭示超微结构特点和生物膜的专业和获得超微结构水平尺寸和空间数据在材料科学和纳米技术产品的首选方法。

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 ...

fundamental-technical-elements-freeze-fracturefreeze-etch-biological

Universidad San Sebastian

Research

JoVE Journal

Biology

33.2K 次观看.  The University of North Carolina at Chapel Hill. 基本的技术和用于检查通过透射电子显微镜生物标本和纳米材料的冷冻断裂加工的精炼进行说明。这种技术对于揭示超微结构特点和生物膜的专业和获得超微结构水平尺寸和空间数据在材料科学和纳米技术产品的首选方法。 

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

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

We present a protocol for the isolation and freeze-fracturing of extracellular vesicles (EVs) originating from cancerous urothelial cells. The freeze-fracture technique revealed the EVs' diameter and shape and-as a unique feature-the internal organization of the EV membranes. These are of immense importance in understanding how EVs interact with the recipient membranes.

用于细胞外囊泡分析的冻裂-断裂电子显微镜

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular ...

科研

癌症研究

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 ...

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 ...

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

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, ...

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 ...

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 ...

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.