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bioRxiv · 10.64898/2026.07.16.738981

Protein-based, live-cell PAINT microscopy with fluorogenic exchangeable HaloTag ligands

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

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.

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Zhan, T., Gerstner, N. C., Martin, J. G., McCann, J. T., Lynch, M. C., Wu, H.-J., Xu, K., Miller, E. W.. 2026-07-17. Protein-based, live-cell PAINT microscopy with fluorogenic exchangeable HaloTag ligands. https://doi.org/10.64898/2026.07.16.738981

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