冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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’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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Balzers Freeze-fracture/freeze-etch plant | Balzers | BAF400T | |
| Standard buffers | various suppliers | ||
| standard aldehyde fixatives | various suppliers | ||
| sodium dichromate | various suppliers | ||
| sulfuric acid | various suppliers | ||
| Disposable supplies for Platinum/Carbon Evaporation | Technotrade International | ||
| Liquid nitrogen | various suppliers | ||
| Freon | various suppliers | ||
| Disposable supplies for electron microscopy | Electron Microscopy Sciences | ||
| Transmission electron microscope | Carl Zeiss Inc. |
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