通过实时细胞成像与电子显微镜关联技术在高分辨率下分析核结构。

Live CLEM imaging to analyze nuclear structures at high resolution.

作者信息

Haraguchi Tokuko, Osakada Hiroko, Koujin Takako

机构信息

Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe, 651-2492, Japan,

出版信息

Methods Mol Biol. 2015;1262:89-103. doi: 10.1007/978-1-4939-2253-6_6.

DOI:10.1007/978-1-4939-2253-6_6

PMID:25555577

Abstract

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

摘要

荧光显微镜（FM）和电子显微镜（EM）是观察细胞中分子成分的强大工具。FM可以提供活细胞中细胞蛋白质和结构的时间信息。EM提供固定细胞中细胞结构的纳米级分辨率图像。我们将FM和EM相结合，开发了一种新的相关光电子显微镜（CLEM）方法，称为“活细胞CLEM”。在这种方法中，首先使用荧光显微镜（FM）在活细胞中观察感兴趣的特定分子的动态行为，然后使用电子显微镜（EM）观察同一细胞中的细胞结构。图像采集后，比较FM和EM图像，使荧光图像与使用EM获得的细胞结构高分辨率图像相关联。由于这种方法能够在高分辨率下分析特定细胞结构背景下涉及感兴趣的特定分子的动态事件，因此对于研究包括核体在内的核结构很有用。在这里，我们描述了可应用于研究哺乳动物细胞核结构的活细胞CLEM。