Analysis Methods for Sensitized Emission
Sensitized emission analysis is available when the image has at least three channels (donor, acceptor, FRET; corresponding measured signals are D, A, and F). The measured intensities are first corrected for crosstalk, for which the following factors are considered: FD/DD; AD/FD; DA/AA; DA/FA; FA/AA. The capital letters refer to channels and subscripts refer to the D and A dyes; for instance, FD/DD is the F/D signal ratio measured for a donor-only sample. In addition, unequal detection efficiencies of the donor and FRET channels are considered (G factor).
In all cases the FRET values are calculated for every pixel that is considered “valid” and then the mean value is calculated and presented in the table.
FC or Youvan Method
Youvan et al., Biotechnology et alia 3, 1 (1997)
Fc = F - D*(FD/DD) - A*(FA/AA)
Displays the Fc image with intensities converted from the FRET index calculated for each pixel using the Youvan method. This method assumes that the signal recorded in the FRET channel is the sum of real FRET signal overlaid by donor crosstalk and acceptor signal induced by direct (donor) excitation. There is no correction for donor and acceptor concentration levels and as a result the FRET values tend to be higher for areas with higher intensities.
FRETN or Gordon Method
Gordon et al., Biophys J 74, 2702 (1998)
FRETN = Fcorr/(Dcorr*Acorr)
Displays the FRET image with intensities converted from the FRET index calculated for each pixel using the Gordon method. This method calculates a corrected FRET value and divides by concentration values for donor and acceptor. This method attempts to compensate for variances in fluorochrome concentrations but overdoes it. As a result, cells with higher molecular concentrations report lower FRET values.
See also
N-FRET or Xia Method
Xia and Liu, Biophys J 81, 2395 (2001)
NFRET = Fcorr/(Dcorr*Acorr)1/2
Displays an N-FRET image with intensities converted from the FRET index calculated for each pixel using the Xia method. This method is like the Gordon method with the difference that the square root of donor and acceptor concentration is used for concentration compensation. The resulting image is properly corrected for variances in the fluorochrome concentration.
See also
Crosstalk Correction for Gordon and Xia Methods
For the Gordon and Xia methods, crosstalk correction is the same.
Acorr = (A – F*AD/FD) / (1 – FA/AA*AD/FD)
Fcorr = (F – D*FD/DD – Acorr*(FA/AA – FD/DD*DA/AA)) / (1 – DA/FA*FD/DD) / G
Dcorr = D + Fcorr*(1 – G*DA/AA) – Acorr*DA/AA