Department of Microbiology and Immunology, The University of Western Ontario, London, ON, Canada.
Robarts Research Institute, London, ON, Canada.
Methods Mol Biol. 2022;2440:57-73. doi: 10.1007/978-1-0716-2051-9_3.
Live cell microscopy has become a common technique for exploring dynamic biological processes. When combined with fluorescent markers of cellular structures of interest, or fluorescent reporters of a biological activity of interest, live cell microscopy enables precise temporally and spatially resolved quantitation of the biological processes under investigation. However, because living cells are not normally exposed to light, live cell fluorescence imaging is significantly hindered by the effects of photodamage, which encompasses photobleaching of fluorophores and phototoxicity of the cells under observation. In this chapter, we outline several methods for optimizing and maintaining long-term imaging of live cells while simultaneously minimizing photodamage. This protocol demonstrates the intracellular trafficking of early and late endosomes following phagocytosis using both two and three dimensional imaging, but this protocol can easily be modified to image any biological process of interest in nearly any cell type.
活细胞显微镜技术已成为探索动态生物学过程的常用技术。当与感兴趣的细胞结构的荧光标记物或感兴趣的生物活性的荧光报告物结合使用时，活细胞显微镜技术能够精确地、在时间和空间上解析正在研究的生物学过程。然而，由于活细胞通常不暴露于光线下，活细胞荧光成像受到光损伤的显著影响，包括荧光团的光漂白和观察细胞的光毒性。在本章中，我们概述了几种优化和维持活细胞长期成像的方法，同时最大限度地减少光损伤。该方案使用二维和三维成像演示了吞噬作用后早期和晚期内体的细胞内转运，但该方案可以轻松修改，以在几乎任何细胞类型中对任何感兴趣的生物过程进行成像。
