Advanced Fluorescence Microscopy Methods to Study Dynamics of Fluorescent Proteins In Vivo

Fluorescent proteins are standard tools for addressing biological questions in a cell biology laboratory. The genetic tagging of protein of interest with fluorescent proteins opens the opportunity to follow them in vivo and to understand their interactions and dynamics. In addition, the latest advances in optical microscopy image acquisition and processing allow us to study many cellular processes in vivo. Techniques such as fluorescence lifetime microscopy and hyperspectral imaging provide valuable tools for understanding fluorescent protein interactions and their photophysics. Finally, fluorescence fluctuation analysis opens the possibility to address questions of molecular diffusion, protein-protein interactions, and oligomerization, among others, yielding quantitative information on the subject of study. This chapter will cover some of the more important advances in cutting-edge technologies and methods that, combined with fluorescent proteins, open new frontiers for biological studies.