Zhan, T.; Gerstner, N. C.; Martin, J. G.; McCann, J. T.; Lynch, M. C.; Wu, H.-J.; Xu, K.; Miller, E. W.

We report a generalizable method for single-molecule localization super-resolution microscopy in living cells. Live-cell protein-based PAINT (points accumulation for imaging in nanoscale topography) is achieved by pairing exceptionally fluorogenic bis-trifluoromethyl rhodamine (BF) dyes with reversible HaloTag ligands. This far-red small molecule update to protein-based PAINT is readily incorporated into existing super-resolution microscopy workflows: pairing with photoactivatable fluorescent proteins further enables simultaneous two-color live-cell super-resolution microscopy and single-molecule diffusivity mapping (SMdM).