Positive-Type ReversiblyPhotoswitching Red FluorescentProtein for Dual-Color Superresolution Imaging with Single Light Exposurefor Off-Switching

Positive-type reversibly photoswitching fluorescent proteins (p-rsFPs) transition to a bright on-state upon light exposure for fluorescence excitation and to a dark off-state under a different wavelength. p-rsFPs are widely used in superresolution (SR) imaging techniques, offering simplified observation procedure and enhanced biocompatibility. Although some green p-rsFPs possess adequate photoproperties for SR imaging, all red p-rsFPs (p-rsRFPs) to date exhibit suboptimal properties, limiting the color palette for multiplexed SR imaging. Here, we present a p-rsRFP, rsZACRO, with 3.0-fold brighter fluorescence, 5.3-fold faster off-switching, and 1.5-fold higher on/off contrast than rsCherry, a conventional representative p-rsRFP. Using rsZACRO with superresolution polarization demodulation/on-state polarization angle narrowing (SPoD-OnSPAN), we successfully demonstrated SR imaging in the red spectrum and dual-color SR imaging with a single light for off-switching, visualizing vimentin intermediate filaments and actin filaments at higher spatial resolution than the diffraction limit of light in a living mammalian cell.