Ratiometric fluorescence imaging of dual bio-molecular events in single living cells using a new FRET pair mVenus/mKOκ-based biosensor and a single fluorescent protein biosensor

► We established a novel combination of fluorescent protein-based biosensors for dual-parameter ratiometric imaging in single living cells. ► Using this approach, we achieved simultaneous imaging of Src/Ca2+ signaling and GSH redox potential in single living cells, which was previously unattainable. ► Furthermore, we provided evidence that EGF-induced Src signaling was negatively regulated by H2O2 via its effect on GSH-based redox system.