Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy
Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy
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DOI：
10.1016/j.jsb.2012.12.004
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发表时间：
2013-03-01
影响因子：
3
通讯作者：
Koster, A. J.
中科院分区：
文献类型：
--
作者：
Faas, F. G. A.;Barcena, M.;Koster, A. J.
相关光学和电子显微镜是一种越来越受欢迎的技术，以各种分辨率水平研究复杂的生物系统。荧光显微镜可用于扫描大面积以定位感兴趣的区域，然后通过电子显微镜进行分析，以从选定的视场以nm级分辨率获得形态和结构信息。以前，室温相关显微镜的综合方法进行了描述。在一台仪器中结合使用光学和电子显微镜，大大简化了样品处理，避免了繁琐的实验开销，简化了两种模式之间的导航，并提高了图像相关性的成功率。在这里，一个集成的方法，在低温条件下的相关显微镜。其相对于室温方法的优点包括保护生物样本的天然水合状态，保存荧光信号而没有由于重原子染色而淬灭的风险，以及减少光漂白。冷冻集成光和电子显微镜的潜力被证明为活菌的检测，在体外聚合的微管的研究，小鼠胚胎成纤维细胞中的线粒体的本地化，并在哺乳动物细胞内的病毒诱导的细胞内膜修饰的搜索。(c)2012 Elsevier Inc. All rights reserved.
Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. (c) 2012 Elsevier Inc. All rights reserved.