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.
Keywords: Acquisition parameters; Fluorescence microscopy; Intracellular trafficking; Live cell imaging; Phagocytosis; Photobleaching; Photodamage; Phototoxicity; Time-lapse; z-stack.
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