Two-color RESOLFT nanoscopy with green and red fluorescent photochromic proteins

Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously.