Imaging the redox states of live cells with the time-resolved fluorescence of genetically encoded biosensors

Redox environments in cells influence many important physiological and pathological processes. In this study, the time-resolved fluorescence of a recently reported thiol redox-sensitive sensor based on vertebrate fluorescent protein UnaG, roUnaG, was studied, along with the application of the time-resolved fluorescence of roUnaG to image the redox states of the mitochondria, cytoplasm, and nucleus in live cells. Time-resolved fluorescence images of roUnaG clearly demonstrated that potent anticancer compound KP372-1 induced extreme oxidative stress. A more stressful redox state observed in activated macrophages further demonstrated the validity of roUnaG with time-resolved fluorescence. For comparison, time-resolved fluorescence images of four other frequently used redox biosensors (roGFP1, HyPer, HyPerRed, and rxRFP) were also captured. The time-resolved fluorescence allows an intrinsically ratiometric measurement for biosensors with one excitation wavelength and provides new opportunities for bioimaging.