自噬相关结构的三色同步实时成像

Three-Color Simultaneous Live Imaging of Autophagy-Related Structures.

作者信息

Ueda Hiroyuki, Kunitaki Ouin, Hamasaki Maho

机构信息

Department of Genetics, Osaka University Graduate School of Medicine, Osaka, Japan.

Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

出版信息

Methods Mol Biol. 2019;1880:223-230. doi: 10.1007/978-1-4939-8873-0_14.

DOI:10.1007/978-1-4939-8873-0_14

PMID:30610700

Abstract

Simultaneous live cell imaging of multiple proteins helps to analyze mobility and interactions among proteins over time. Since autophagosomes depend on other organelles for their formation, it is necessary to observe this process with multiple fluorsphores to mark multiple organelles and the autophagosomes. To do so, we set up three cameras on one microscope to be able to acquire three colors at the same time. Here we describe the setup using a Yokogawa spinning disk confocal microscope (CSU-W1) with Andor TuCam system attaching 3 × Zyla 4.2 CMOS cameras (Andor) and detail the method for acquiring live images.

摘要

对多种蛋白质进行同步活细胞成像有助于分析蛋白质随时间的移动性和相互作用。由于自噬体的形成依赖于其他细胞器，因此有必要使用多种荧光团来标记多个细胞器和自噬体，从而观察这一过程。为此，我们在一台显微镜上设置了三台相机，以便能够同时采集三种颜色的图像。在此，我们描述了使用配备Andor TuCam系统的横河旋转盘共聚焦显微镜（CSU-W1）的设置方法，该系统连接了3台Zyla 4.2 CMOS相机（Andor），并详细介绍了获取活细胞图像的方法。