From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 153

Stokes shift

Absorption Emission

Wavelength

Figure 5.6 Illustration of the Stokes shift between the absorption and emission spectra.

The excited molecules (M * ) could be de-excited by radiative ( K r )or
nonradiative ( K nr ) processes whose intersystem transfer as FRET ( K T ). In
classical kinetics, the rate of disappearance of excited molecules is expressed
by the differential equation

dM ½
d t ¼

M ð K r þ K nr Þ ½ ½ 5 : 5

Integration of this equation yields the time evolution of the concentra-
tion of the excited molecules [M * ] (see Fig. 5.4 ).

M M 0 e t= t

ð Þ

½ ¼ ½ ½ 5 : 6

1
K r þ

With t ¼ ½ 5 : 7

K nr

[M * ] 0 is the concentration of the excited molecules at time 0, which re-
sults from light excitation. The fluorescence lifetime t is in the range of 10 -9 s
(0.5-20 ns for commonly used fluorescence transitions). t describes the av-
erage time for which a molecule stays in its excited state before emitting a
photon. 7 The resonance transfer or FRET ( K T ) is included in nonradiative
deactivation pathways ( K nr ). It can be defined as the difference between the
rates K nr of the donor only and K nr of the donor in the presence of the