FRET efficiency and maturity of ShadowR in tandem fluorescent proteins. (a) A schematic drawing of the constructs used to evaluate the FRET efficiency and fraction of the mRuby2 or mScarlet undergoing FRET. (b) Representative fluorescence lifetime images of the tandem proteins in HeLa cells; the images were taken at 1000-nm two-photon excitation. Because the expression level of mRuby2-Ultramarine and mScarlet-Ultramarine was low, we used different laser powers for each condition (5 mW for mRuby2-Ultramarine, 4 mW for mRuby2-ShadowR, 3 mW for mScarlet-Ultramarine, 2 mW for mScarlet-ShadowR). Scale bar, 50 µm. (c,e) Quantification of FRET efficiency of the tandem proteins. The fluorescence lifetime over the whole image was used for the analysis (See Materials and Methods). The number of images used for the analysis are indicated in the figure. Each image contains 4–12 cells, and the data are presented as mean ± SEM. Asterisks denote statistical significance (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant). (d,f) A comparison of the fraction of mRuby2 or mScarlet fluorescent protein undergoing FRET (chromophore maturation efficiency of Ultramarine or ShadowR) analyzed in individual cells; data were plotted in the descending order. The FRET fraction is directly related to the maturation efficiency of an acceptor, i.e., Ultramarine (d) or ShadowR (f). Means ± SD are also plotted on the right (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant). The number of samples (n) and mean ± SD are also indicated. (g) Comparison of E. coli expressing Ultramarine, ShadowR, or dUltramarine2. Purple colonies indicate that the E. coli cells express the respective chromoproteins. After transformation, the cells were incubated for 16 hr at 32 °C and imaged.