Live-cell PAINT microscopy of protein targets via fluorogenic exchangeable HaloTag ligands

We report a generalizable method for single-molecule localization super-resolution microscopy in living cells. Live-cell PAINT (points accumulation for imaging in nanoscale topography) and single-molecule diffusivity mapping (SMdM) of intracellular protein targets are achieved by pairing exceptionally fluorogenic bis-trifluoromethyl rhodamine (BF) dyes with reversible HaloTag ligands and widely available HaloTag fusion proteins. This far-red small-molecule update to protein-based PAINT is readily incorporated into existing super-resolution microscopy workflows: wash-free staining and imaging are achieved for cell lines and primary neuron cultures for diverse protein targets. Pairing with photoconvertible fluorescent proteins further enables simultaneous two-color live-cell super-resolution microscopy and SMdM.