Figure 5
From: ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
FRET efficiency and maturity of ShadowY in tandem fluorescent proteins. (a) A schematic drawing of the tandem fluorescent protein used to evaluate the FRET efficiency and fraction of the donor fluorescent protein undergoing FRET (chromophore maturation efficiency) for ShadowY. (b,d,f) Comparison of FRET efficiency of the tandem fluorescent proteins. The fluorescence lifetime decay curve averaged over the whole image was used for the analysis (See Materials and Methods). The number of images used for the analysis is 10–13. Each image contains 8–15 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). (c,e,g) A comparison of the fraction of donor fluorescent protein undergoing FRET analyzed in individual cells and data was plotted in the descending order. The FRET fraction is directly related to the maturation efficiency of an acceptor, i.e., sREACh or ShadowY. 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 in figures.
