Schmitz Michael H A, Gerlich Daniel W
Institute of Biochemistry, ETH Zurich, Zurich, Switzerland.
Methods Mol Biol. 2009;545:113-34. doi: 10.1007/978-1-60327-993-2_7.
Fluorescence live microscopy is a powerful technique to study complex cellular dynamics such as cell division. The availability of fluorescent markers based on GFP fusion proteins for virtually any cellular structure allows efficient visualization of specific processes, and the combination of different fluorophores can be used to study their coordination. In this chapter, we present methods for automated live cell microscopy to study mitotic gene function systematically and in high throughput. In particular, we provide protocols for efficient generation of fluorescent reporter cell lines stably expressing combinations of cellular markers, and provide detailed guidelines for optimizing imaging protocols for automated long-term live microscopy.
荧光活细胞显微镜术是研究诸如细胞分裂等复杂细胞动力学的强大技术。基于GFP融合蛋白的荧光标记物几乎可用于任何细胞结构，这使得特定过程能够高效可视化，并且不同荧光团的组合可用于研究它们的协同作用。在本章中，我们介绍了用于自动活细胞显微镜术的方法，以系统地、高通量地研究有丝分裂基因功能。特别是，我们提供了有效生成稳定表达细胞标记物组合的荧光报告细胞系的方案，并为优化自动长期活细胞显微镜术的成像方案提供了详细指南。
