Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Nat Commun. 2020 Jan 14;11(1):273. doi: 10.1038/s41467-019-14067-4.


We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bilirubin / metabolism*
  • Fluorescence
  • Green Fluorescent Proteins / metabolism*
  • Kinetics
  • Ligands
  • Light
  • Microscopy, Fluorescence
  • Photochemical Processes
  • Protein Binding
  • Single Molecule Imaging / methods*


  • Ligands
  • Green Fluorescent Proteins
  • Bilirubin