Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein

Mol Biol Cell. 2015 Jun 1;26(11):2054-66. doi: 10.1091/mbc.E14-10-1473. Epub 2015 Apr 15.


We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cytoplasm
  • Fluorescent Dyes*
  • Green Fluorescent Proteins*
  • HEK293 Cells
  • Humans
  • Luminescent Proteins*
  • Microscopy, Fluorescence, Multiphoton
  • Molecular Sequence Data
  • Potoroidae
  • Protein Binding
  • Protein Multimerization*
  • Sequence Alignment
  • Spectrometry, Fluorescence / methods*


  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins