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as in (2), provided by the second term in (4). Thus, this formula can be applied to
almost any dye, but caution is advised when used for FPs.
A speciality of FPs is that e
abs
(n) is related to 1 mole proteins but not to 1 mole of
chromophoric units. The value neglects, on the one hand, that not every protein has a
fully completed chromophore, i.e., incomplete chromophore maturation [
31
]. On the
other hand, equilibria between different chromophore forms, e.g. the neutral and the
anionic chromophore forms in wt-GFP, are also neglected. Mutations in FPs which do
not alter the chromophore mainly influence these two parameters. Hence, a correction
factor x might be introduced: x should denote the fraction of proteins of a certain
mutant, which effectively contribute to the
S
0
!
S
1
transition under investigation (5).
ð
4
m
e
e
0
c
e
2
ln 10
x
f
12
¼
e
abs
ðÞ
dn
:
(5)
N
A
The meaning of (4) is that, as
f
12
and both terms in the parentheses are constant,
the integral over the frequency is also constant. In other words, the area under the
absorption spectrum is constant for a specific transition of a certain chromophore
form. A higher e
max
in an absorption spectrum results in a reduced width of the
absorption band. In terms of quantum mechanics, the shape of the excitation as well
as the emission spectrum is modulated by the Franck-Condon factors
f
FC
(see also
Sect.
1.4
). However, the natural lifetime t
rad
of the
S
1
state is virtually constant if
the spectral changes of the same transition upon mutation are minor. This relation
was experimentally verified in a series of GFP and YFP variants; it turned out that
the whole variation of
A
21
is less than 10% [
21
]. It is worth to emphasize that an
accurate calculation of the extinction coefficients or the oscillator strength is
absolutely necessary for a correct and unbiased determination of t
rad
(2).
1.4 Relevant Radiationless Processes
The decay of the upper state of a TLS like in Fig.
1
obeys first-order kinetics (6)
with
A
21
as the decay rate constant. Sometimes,
k
rad
is used instead of
A
21
.
d
S
½
d
t
¼
A
21
S½!
S½ð
t
Þ¼
S½
exp
ð
A
21
t
Þ:
(6)
The purely radiative lifetime, which could be measured if no fluorescence
quenching took place, is t
rad
¼
A
1
21
. Fluorescence lifetime measurements, however,
always lead to t
fl
, which is shorter than t
rad
. A faster decay of the excited state
S
1
is
the result of additional decay channels from the excited state
S
1
to the electronic
ground state
S
0
. In the kinetic description, these exclusively non-radiative channels
are accounted for by introducing additional rate constants. They are subsumed by
k
IC
, the rate constant for internal conversion (7).
d
S
½
d
t
¼
1
t
Fl
¼
ð
A
21
þ
k
IC
Þ
S½!
A
21
þ
k
IC
:
(7)
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