Probing subtle fluorescence dynamics in cellular proteins by streak camera based fluorescence lifetime imaging microscopy

We report the cell biological applications of a recently developed multiphoton fluorescence lifetime imaging microscopy system using a streak camera (StreakFLIM). The system was calibrated with standard fluorophore specimens and was shown to have high accuracy and reproducibility. We demonstrate the applicability of this instrument in living cells for measuring the effects of protein targeting and point mutations in the protein sequence, which are not obtainable by conventional intensity-based fluorescence microscopy methods. We discuss the relevance of such time-resolved information in quantitative energy transfer microscopy and in measurement of the parameters that characterize the intracellular physiology.