Binding of ECFP-hnH to its RNA binding site by FRET-FLIM in situ. (A) Lifetime of ECFP fluorescence was measured for ECFP-p80 as a negative control and ECFP-p80 coexpressed with EYFP-p80 as a positive control. Lifetimes within cells are shown in false colors: (blue) short lifetime; (red) long lifetime. Notice shortening of ECFP fluorescence lifetime, which is indicative of FRET. (B) ECFP-hnH and MS2-EYFP were coexpressed with constructs containing different binding sites, and the ECFP fluorescence lifetime was measured for each construct. pRed-M4x served as a negative control. ECFP fluorescence, lifetime heat-maps, and histograms of lifetime distributions are shown. (C) Quantification of lifetimes shown in A and B. Decrease of lifetimes indicates FRET between ECFP and EYFP constructs. In all cases, mean lifetimes of the biexponential fit are shown. The average and SEM are shown (SDs are indicated in Table 1), and the number of assayed cells is indicated within the bars.
