Figure 4
From: ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
Performance of ShadowY in LOV2 FRET sensors in HeLa cells. (a) A schematic of a conformational change of the light-sensitive LOV2 FRET sensors. (b) Representative fluorescence lifetime images of mEGFP-LOV2-ShadowY after blue light illumination for 2 seconds at 35 mW/cm2. The scale bar is 50 µm. (c) An averaged time course of fluorescence lifetime changes in response to blue light illumination. The number of cells analyzed is 70 for mEGFP–sREACh and 73 for mEGFP–ShadowY. The data are presented as mean ± SEM. (d) An averaged time course of fluorescence lifetime changes in response to blue light illumination. The number of cells analyzed is 79 for Clover–sREACh, 76 for Clover–ShadowY, 74 for CloverT153M–ShadowY, and 73 for CloverT153MF223R–ShadowY. The data are presented as mean ± SEM. (e,f) The fluorescence lifetime changes at 20 sec after blue light illumination were quantified using the data presented in (c,d), respectively. The data are presented as mean ± SEM. Asterisks denote statistical significance (p < 0.05, analysis of variance [ANOVA] followed by Scheffé’s post hoc test). N. S. = not statistically significant. (g–l) The lifetime changes in individual HeLa cells before and after light illumination (the same dataset as in panels c and d). The basal fluorescence lifetime (averaged over −1.3 to 0 min) of individual cells is plotted in the descending order (black) along with the corresponding fluorescence lifetime values (at 20 sec) after blue-light illumination (red). The data are also presented as mean ± SD on the right. The number of samples (n) is indicated in respective panels.
