Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes

Biophys J. 2001 Oct;81(4):2395-402. doi: 10.1016/S0006-3495(01)75886-9.


Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / analysis*
  • Bacterial Proteins / chemistry
  • Cell Membrane / chemistry
  • Cells, Cultured
  • Energy Transfer
  • Fluorescence
  • Green Fluorescent Proteins
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / chemistry
  • Membrane Proteins / analysis*
  • Membrane Proteins / chemistry
  • Microscopy, Confocal
  • Microscopy, Fluorescence / methods*
  • Nerve Tissue Proteins / analysis*
  • Nerve Tissue Proteins / chemistry
  • Qa-SNARE Proteins
  • Rats
  • Recombinant Fusion Proteins / analysis*
  • Recombinant Fusion Proteins / chemistry
  • Reproducibility of Results
  • Spectrometry, Fluorescence
  • Synaptosomal-Associated Protein 25


  • Bacterial Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Qa-SNARE Proteins
  • Recombinant Fusion Proteins
  • Snap25 protein, rat
  • Synaptosomal-Associated Protein 25
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins