Schauflinger Martin, Villinger Clarissa, Walther Paul
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Methods Mol Biol. 2013;1064:227-37. doi: 10.1007/978-1-62703-601-6_16.
In this paper we show how to obtain a three-dimensional model of virus-infected cells by serial sectioning of resin embedded samples and transmission electron microscopic imaging. The method bases on sample fixation by high pressure freezing and processing by freeze substitution with the goal to preserve the structures of interest close to the natural state, as previously described (Walther et al., High pressure freezing for scanning transmission electron tomography analysis of cellular organelles. In: Mossman BT, Taatjes DJ (eds) Cell imaging techniques, vol 931, Methods in molecular biology. Humana Press, Totowa, NJ, pp 525-535, 2013). Advantages of serial sectioning compared to that of other tomographic methods are as follows: No special and expensive additional equipment is required. Relatively large volumes, such as whole cells, can be three-dimensionally reconstructed in a reasonable amount of time. Serial sectioning is a non-destructive method; the sections can be stored, re-imaged, or processed for immunogold labeling when more specific data are requested or when new scientific questions are raised (e.g., higher magnifications, protein distributions). We have recently used this method to obtain a three-dimensional model of the complete assembly complex of an HCMV infected cell, which allowed a detailed insight into this virally induced compartment (Schauflinger et al., Cell Microbiol 15(2):305-314, 2013).
在本文中，我们展示了如何通过对树脂包埋样本进行连续切片以及透射电子显微镜成像来获得病毒感染细胞的三维模型。该方法基于高压冷冻固定样本并通过冷冻置换进行处理，目的是将感兴趣的结构保持在接近自然状态，如先前所述（Walther等人，《用于细胞器扫描透射电子断层扫描分析的高压冷冻》。载于：Mossman BT、Taatjes DJ（编）《细胞成像技术》，第931卷，分子生物学方法。人类出版社，托托瓦，新泽西州，第525 - 535页，2013年）。与其他断层扫描方法相比，连续切片的优点如下：不需要特殊且昂贵的额外设备。相对较大的体积，如整个细胞，可以在合理的时间内进行三维重建。连续切片是一种非破坏性方法；当需要更具体的数据或提出新的科学问题时（例如，更高的放大倍数、蛋白质分布），切片可以保存、重新成像或进行免疫金标记处理。我们最近使用这种方法获得了人巨细胞病毒感染细胞完整组装复合体的三维模型，这使得能够详细了解这个病毒诱导的区室（Schauflinger等人，《细胞微生物学》15(2):305 - 314，2013年）。
