From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 161

The apparent transfer efficiency (E) can be written as

1

R 0
R 0 þ

Pr ðÞ

E ¼

r 6 d r

½ 5 : 25

0

Bulk measurements of FRET efficiency by intensity-based methods cannot
distinguish between an increase in FRET efficiency (i.e., coupling efficiency)
and an increase in FRET population (concentration of FRET species) since
both parameters are not resolved. FRET measurements based on the
analysis of the donor fluorescence lifetime may resolve this problem with
multiexponential decay models. (7,23) The assumption that interacting and
noninteracting populations are present allows the determination of both the
efficiency of interaction and the fractional population of the interacting
molecules. In the first instance, the presence/absence of FRET is determined
by fitting the experimental data to a single-exponential decay. 6 Sufficient
reduction in the measured lifetime indicates the existence of FRET.
Additional analysis is subsequently applied to determine the source of
lifetime reduction. In this case, a biexponential fluorescence decay model
applied to the data allows the determination of the fluorescence lifetimes of
noninteracting and interacting subpopulations or two distinct levels of
interaction in case of many biosensors. In time-domain measurements (see
next section), data may be fitted by iterative convolution with

( )

X

a i e t= t i IðÞ¼ IRF ðÞ Offset þ ½ 5 : 26

i

where IRF( t ) is the instrumental response and Offset is the baseline, t i is the
lifetime of interacting or noninteracting populations, and a i is the the pre-
exponential factor relating to the absolute species concentration ( Fig. 5.10 ). 24

3.7. Conclusion on FRET principles and applications
It is difficult to obtain quantitative determination of labeled interacting mol-
ecules from steady-state images. The fluorescence intensity does not only de-
pend on the FRET efficiency but also on the unknown local concentration of
dyes. Up to eight measurements at different excitation wavelengths and in dif-
ferent emission wavelength bands can be used to obtain calibrated FRET re-
sults from steady-state data. 7 In lifetime data, however, FRET shows up as a
dramatic decrease of the donor lifetime. 6 Initially, qualitative FRET results can
be obtained by fitting decay curves with a single-exponential approximation.