From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 160

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

0 0.7 R 0 R 0

Distance

1.4 R 0

Figure 5.9 FRET efficiency as a function of the distance between the donor and the
acceptor. The FRET efficiency is 50% when the distance is equal to the Förster distance
( R 0 ). FRET efficiencies ranging from 10% to 90% correspond to distances between

fluorophores of 1.4 R 0 and 0.7 R 0 , respectively.

t DA
t D

E ¼ 1 ½ 5 : 24

The FRET kinetic measurement can be performed by the calculation of
the ratio ( R ) between the two stationary states' kinetics. R is classically used
for measurements by FRET-based biosensors (see next section). 22
Both Eqs. (5.23) and (5.24) are applicable only to donor/acceptor pairs
that are separated by a fixed distance. However, single-exponential decays
are rare in biology.
The mean lifetime t mean defined by Eq. (5.15) has been largely used in
FRET experiments. 8-11 This mean lifetime is then equivalent to the area of
the fluorescence intensity decay, which is related to the FRET efficiency E .
However, the mean lifetime does not correspond to the correct average
lifetime, which is defined by Eq. (5.12) .

3.6. FRET measurements of molecular populations
In FRET analysis, particularly for biosensors, two elements must be usually
considered: the interacting fluorophore population and the FRET efficiency.
The distance distribution density function P ( r ) describes the probability
of finding the specific donor/acceptor pair separation.