Combining TIR and FRET: From Fluorescence Microscopy to a Multi-well Reader System

摘要

In view of a pharmacological test system with living cells we examined F?rster Resonance Energy Transfer(FRET) between fluorescent proteins upon Total Internal Reflection (TIR) of a laser beam. We measured inparticular HeLa cells expressing membrane associated EGFR-CFP and Grb2-YFP prior to and subsequent tostimulation with the epidermal growth factor EGF. In addition, HeLa cells expressing a membrane associatedGreen Fluorescent Protein (HeLa hFR-GPI-GFP) were incubated with the energy acceptor Nile Red for furthervalidation. Measurements range from fluorescence microscopy to Fluorescence Lifetime Imaging (FLIM) of alarger number of samples in a TIR reader.