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