Heuser John E
Department of Cell Biology, Washington University School of Medicine, St. Louis, MO, USA.
J Electron Microsc (Tokyo). 2011;60 Suppl 1(Suppl 1):S3-29. doi: 10.1093/jmicro/dfr044.
The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future.
1961年，摩尔引入了巴尔泽斯冷冻断裂机，这对电子显微镜的发展产生了比他所能想象的更大的影响。冷冻断裂技术最初是为了规避传统超薄切片技术的风险，同时提供细胞膜独特的正面视图而设计的，事实证明，它对于形成关于生物膜如何组织的现代概念至关重要，并证明了膜是由蛋白质漂浮并自组装其中的脂质双层组成。后来，当冷冻断裂技术与避免了摩尔在为其原始发明制备样品时仍需使用的固定和冷冻保护步骤的细胞冷冻方法相结合时，它成为了一种在毫秒时间尺度上捕捉膜动态的手段，从而使人们能够更深入地了解活细胞中生物膜的功能及其静态超微结构。最后，人们意识到，在冷冻断裂后，未固定、未冷冻保护的样品可以进行深度真空蚀刻甚至冷冻干燥，这开辟了一种全新的方法来对细胞除膜之外的所有其他分子成分进行成像，同时也提供了一种强大的手段来成像细胞所有细胞质成分与细胞各种膜之间的相互作用。本综述的目的是概述这些技术发展的历史，描述它们如今在电子显微镜中的应用方式，并提出如何改进它们，以便在未来进一步提高它们在生物电子显微镜中的效用。
