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首页> 外文期刊>Journal of Structural Biology >Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections
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Self-pressurized rapid freezing (SPRF) as a simple fixation method for cryo-electron microscopy of vitreous sections

机译：自加压快速冷冻（SPRF）作为玻璃体切片冷冻电子显微镜的简单固定方法

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摘要

Cryo-electron microscopy of vitreous sections (CEMOVIS) is currently considered the method of choice to explore cellular ultrastructure at high resolution as close as possible to their native state. Here, we apply a novel, easy-to-use and low-cost freeze fixation method for CEMOVIS, avoiding the use of high-pressure freezing apparatus. Cells are placed in capillary metal tubes, which are tightly closed and plunged directly into liquid ethane cooled by liquid nitrogen. In some parts of the tube, crystalline ice is formed, building up pressure sufficient for the liquid-glass transition of the remaining specimen. We verified the presence of vitreous ice in these preparations using CEMOVIS and electron diffraction. Furthermore, different tube materials being less poisonous than copper were established to minimize physiological alterations of the specimen. Bacteria, yeast and mammalian cells were tested for molecular resolution. The quality of results is equivalent to samples prepared by conventional high pressure freezing apparatus, thus establishing this novel method as fast, easy-to-use and low-cost freeze fixation alternative for cryo-EM. (C) 2012 Elsevier Inc. All rights reserved.
机译：玻璃体切片的低温电子显微镜（CEMOVIS）目前被认为是一种以尽可能接近其原始状态的高分辨率探索细胞超微结构的选择方法。在这里，我们为CEMOVIS应用了一种新颖，易于使用且低成本的冷冻固定方法，避免了使用高压冷冻设备。将细胞放置在毛细管金属管中，将其密闭并直接插入液态氮冷却的液态乙烷中。在试管的某些部分，形成了结晶冰，积聚了足够的压力，足以使其余样品发生液-玻璃转变。我们使用CEMOVIS和电子衍射验证了这些制剂中玻璃冰的存在。此外，建立了比铜毒性更小的不同试管材料，以最大程度地减少标本的生理变化。测试了细菌，酵母和哺乳动物细胞的分子分辨率。结果的质量等同于常规高压冷冻设备制备的样品，因此将这种新方法确立为快速，易于使用且低成本的冷冻-EM冷冻固定替代品。 （C）2012 Elsevier Inc.保留所有权利。

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