Recent advances in high-pressure freezing: equipment- and specimen-loading methods.
Recent advances in high-pressure freezing: equipment- and specimen-loading methods.
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高压冷冻的最新进展：设备和样本加载方法。
DOI：
10.1007/978-1-59745-294-6_8
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Muller-Reichert,Thomas
中科院分区：
文献类型：
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作者：
McDonald,KentL;Morphew,Mary;Verkade,Paul;Muller-Reichert,Thomas
本章是麦克唐纳在本书第一卷中首次发布的材料的更新。在这里，我们讨论自该文章发表以来高压冷冻 (HPF) 技术和方法的改进。首先，我们介绍 HPF 的最新创新，即 Leica EM PACT2。该机器与前一章的主题 BAL-TEC HPM 010 高压冷冻机有很大不同。 EM PACT2 是一款较小的便携式机器，并具有可选附件快速传输系统 (RTS)。这种 RTS 可以轻松且可重复地加载样品，并允许进行具有高时间分辨率的相关光学和电子显微镜检查。我们在本文中还更加强调 HPF 样品加载的细节，这被认为是整个过程中最关键的阶段。描述了如何高压冷冻悬浮细胞、附着在基质上的细胞、组织样品或小于 300 μm 的整个生物体以及尺寸大于 300 μm 的组织或生物体的详细程序。我们通过对冷冻替代的简短讨论来结束本文，并为此过程推荐了一些示例方案。
This chapter is an update of material first published by McDonald in the first volume of this book. Here, we discuss the improvements in the technology and the methodology of high-pressure freezing (HPF) since that article was published. First, we cover the latest innovation in HPF, the Leica EM PACT2. This machine differs significantly from the BAL-TEC HPM 010 high-pressure freezer, which was the main subject of the former chapter. The EM PACT2 is a smaller, portable machine and has an optional attachment, the Rapid Transfer System (RTS). This RTS permits easy and reproducible loading of the sample and allows one to do correlative light and electron microscopy with high time resolution. We also place more emphasis in this article on the details of specimen loading for HPF, which is considered the most critical phase of the whole process. Detailed procedures are described for how to high-pressure freeze cells in suspension, cells attached to substrates, tissue samples, or whole organisms smaller than 300 μm, and tissues or organisms greater than 300 μm in size. We finish the article with a brief discussion of freeze substitution and recommend some sample protocols for this procedure.