• CFP;
  • concentration ratio;
  • E;
  • FRET;
  • sensitized emission;
  • YFP


Sensitized emission FRET detection method based on three-filter fluorescence microscopy is widely used and more suitable for live cell FRET imaging and dynamic protein–protein interaction analysis. But when it is applied to detect two proteins interaction in living cells, this intensity-based detection method is complicated by many experimental factors such as spectral crosstalk and spectral bleed-through and variable donor to acceptor concentration ratio. There are several FRET algorithms developed recently to correct those factors in order to quantitatively gauge and compare FRET signals between different experimental groups. But the algorithms are often difficult to choose when they are applied to certain experiments. In this research, we use c-Fos/c-Jun as a simple hetero-dimer interaction model to quantitatively detect and compare the FRET signals based on the following widely used sensitized emission FRET algorithms: NFRET, FRETN, FR, FRETR, Eapp and EEFF. We optimized the donor to acceptor concentration ratio range for the above FRET algorithms and facilitate their use in accurate FRET signal determination based on the three-filter FRET microscopy.