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PreprintCC BY-NC-NDbioRxiv

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

  • T. Zhan
  • N. C. Gerstner
  • J. G. Martin
  • J. T. McCann
  • M. C. Lynch
  • H.-J. Wu
  • K. Xu
  • E. W. Miller1
  1. 1University of California, Berkeley
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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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Cite

BibTeXRIS
BibTeX
@misc{zhan2026livecell,
  title = {{Live-cell PAINT microscopy of protein targets via fluorogenic exchangeable HaloTag ligands}},
  author = {T. Zhan and N. C. Gerstner and J. G. Martin and J. T. McCann and M. C. Lynch and H.-J. Wu and K. Xu and E. W. Miller},
  howpublished = {bioRxiv},
  year = {2026},
  month = jul,
  doi = {10.64898/2026.07.16.738981},
  url = {https://www.biorxiv.org/content/10.64898/2026.07.16.738981v2},
  note = {CC BY-NC-ND},
}
RIS
TY  - UNPB
TI  - Live-cell PAINT microscopy of protein targets via fluorogenic exchangeable HaloTag ligands
AU  - T. Zhan
AU  - N. C. Gerstner
AU  - J. G. Martin
AU  - J. T. McCann
AU  - M. C. Lynch
AU  - H.-J. Wu
AU  - K. Xu
AU  - E. W. Miller
PY  - 2026
DA  - 2026/07/16
PB  - bioRxiv
DO  - 10.64898/2026.07.16.738981
UR  - https://www.biorxiv.org/content/10.64898/2026.07.16.738981v2
AB  - 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.
ER  -