From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 151

A

S 1

K nr

Intersystem crossing
collision,...

K T

E ex E em

E tr

K r

S 0

Excitation Fluorescence

FRET donor FRET acceptor

B

M+pha M* M+phe

K r

K nr

M+ D s

Figure 5.4 ( A) Jablonski diagram. S 0 is the fundamental energy level of the electron, S 1
is the first exited level, and the small lines represent the vibrational energy levels. E ex is
the energy of excitation, E em is the energy of fluorescence emission, K r is the radiative
de-excitation rate, and K nr is the nonradiative de-excitation rate. (B) Scheme of the
molecular equilibrium occurring between the absorption and emission processes where
M is the fluorophore (molecule), M* is the excited state of themolecule, pha is the photon
absorbed, phe is the photon emitted, D S is the entropy, k r is the radiative de-excitation
rate, and k nr is the nonradiative de-excitation rate.

where e(l) is the molar absorption coefficient (L mol -1 cm -1 ) and l (cm) is
the absorption path length (sample thickness) of the sample ( Fig. 5.5 ).
The absorbance reflects the probability of a population of fluorophores to
jump to an excited state under the effect of an incident photon at the wave-
length l.
The absorption coefficient a(l) is the absorbance divided by the optical path
length ( l ) in the medium:

,

A ðÞ
l

1
l

I 0
I

I 0 e a ðÞl a ðÞ¼ ¼ log I ¼ ½ 5 : 3