Figure 4. Performance of ShadowG in a FRET sensor based on the LOV2 in HeLa cells.

(a) A schematic of a conformational change in the light-sensitive LOV2 domain. XFP denotes mCherry, sREACh, or ShadowG. (b) Representative fluorescence lifetime images of mEGFP-LOV2-XFP after illumination with blue LED light. Two-photon excitation at 920 nm was used for the excitation of mEGFP. The scale bar is 100 μm. (c) An averaged time course of fluorescence lifetime changes in response to illumination with blue light (closed circles) and without illumination (open circles). The number of cells analyzed is 60 for mCherry, 67 for sREACh, and 65 for ShadowG. In the no-light control, the number of cells analyzed is 26 for mCherry, 25 for sREACh, and 25 for ShadowG. The data are presented as mean ± SEM. (d) The fluorescence lifetime changes at 20 sec after blue light illumination. 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). (e,f) The conformational change of mEGFP-LOV2-XFP in individual HeLa cells after illumination with blue light (the same data set as in panel c). Colored lines represent the response signals from individual cells and the black circles indicate an averaged time course. The data are presented as mean ± SEM. (h–j) 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 lifetimes (at 20 sec) after blue light illumination (red). The data from (e–g) are used in (h–j), respectively. The data are also presented as mean ± SD on the right. Asterisks denote statistical significance (p < 0.05, t test).