Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing

J Microsc. 2004 Aug;215(Pt 2):162-73. doi: 10.1111/j.0022-2720.2004.01365.x.


Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.

MeSH terms

  • Algorithms
  • Animals
  • Bacterial Proteins / analysis
  • Cell Line
  • Connexins
  • Fluorescence Resonance Energy Transfer / methods*
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / analysis
  • Microscopy, Confocal / methods
  • Recombinant Proteins / analysis
  • Transfection


  • Bacterial Proteins
  • Connexins
  • DFNA3 protein, human
  • Luminescent Proteins
  • Recombinant Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins