experiments, etc. as more fluorescence photons are generated from the same number
of excitation cycles. An excitation cycle denotes the excitation from S 0 to S 1 with the
subsequent decay back to S 0 .
Longer t Fl are also preferential for analytical purposes. Also time-resolved
anisotropy experiments would benefit from a longer t Fl as the rotation of FPs is
already in the range of tens of nanoseconds [ 41 ]. As stated above, the actual limit
is given by the radiative rate constant A 21 . Hence, a prolongation of t FL can only
be achieved if the extinction coefficient e abs (n) or, more correctly, the oscillator
strength f 12 can be diminished. At present, this can be solely realized with the
incorporation of small, non-canonical chromophores into FPs, where a trade-off
between IC and the
3 dependence of A 21 as limiting factors has to be found.
It should also be emphasized that the total brightness of FPs will suffer from
lowering f 12 due to its connection with the extinction coefficients (5).
Finally, the sensitivity of lifetime measurements due to increased non-radiative
rate constants k j greatly improves as the lifetime change
D t Fl scales with the square
of t Fl (11).
X k i
X k i
d t Fl
d k j ¼
!D t FL t FL 2
t FL ¼
A 21 þ
A 21 þ
k j :
k j , are altered
FRET efficiencies (9), the accelerated decay due to a higher local refractive index
n 0 according to (2).
Examples for the changes of the mentioned rate constants, i.e.,
2 Measurement of Fluorescence Decays
In the following, we shortly overview several experimental methods for the determi-
nation of t Fl . Recently, several monographs on this topic were released [ 39 , 42 , 43 ].
Also, original publications appeared in the past which compare the different methods
in terms of photon economy, acquisition speed, etc [ 44 - 46 ]. Differences in the t Fl can
be used as contrast mechanism, alternative to the emission colour, and allow for
localizing different FPs [ 47 ]. All these more technical aspects are beyond the scope of
this article, which focuses on lifetime variation of FPs.
2.1 Fluorescence Lifetime Measurements
Lifetime information can be obtained from various methods with different lifetime
resolution. The fastest light-induced processes below 1 ps require pump-probe
techniques. Excited-state lifetimes on this timescale lead to negligible few fluores-
cence photons with F Fl <
10 4 . The S 1 state is probed here either by stimulated