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frequently observed. First of all, higher values of the refractive index n 0 of a cell
enhance A 21 [see (2)] and therefore lead to shorter t Fl [ 25 , 41 ]. This explanation is
mostly chosen when a clear dependence on other parameters is lacking, although
experiments show that t Fl in the cytoplasm is not very different from aqueous
solutions [ 50 ]. Second, local changes of the pH-value or the presence of a (yet
unknown) quencher might lead to changes of t Fl . It was believed in the past that the
fluorophore is well shielded from the solvent by the protein barrel. The finding that
especially YFPs are sensitive to anions shows that this assumption is not generally
valid. Indeed, we observed that even zwitterionic buffer molecules such as CAPS
( c -hexyl-aminopropane sulfonic acid) of considerable size can quench t Fl of YFP at
10 mM concentrations by ~10%. Cellular systems contain a wealth of unidentified
metabolites, and it is imaginable that some of these are locally so abundant that they
can act as quenchers. Systematic investigations which address this issue are lacking
so far, but would be of great importance. Third, temperature variation are certainly
an issue [ 35 , 51 ], at least for some mutants.
Finally, some FPs can undergo reversible or irreversible photochemistry, even-
tually with a concomitant colour change. Those processes are the basis of some
recent microscopy applications of FPs allowing for intelligent timing and super-
resolution imaging in cells. Focusing the radiation of 1 mW continuous wave,
visible laser light to a diffraction limited spot can locally generate intensities I exc
up to 1 MW cm 2 . Such high intensities I exc saturate the S 0 !
S 1 transition, i.e.,
more excitation cycles per second ( k 12 ) cannot be performed. In other words, the
first excited state S 1 is populated at least as strongly as the ground state S 0 (12).
Here, k em denotes the number of fluorescence photons per second which can be
obtained from an individual emitter.
k 12
en ex ðÞ
ln 10
I exc
h n exc :
k em ¼
k 12 t Fl F Fl ;
k 12 ¼
1 þ
It is imaginable that excitation into higher excited states can occur under these
conditions. However, in the limit that k 12 reaches A 21 , the observation of rare events
is favoured. Rare events are processes with quantum yields below 0.1% and are
detectable in cuvette experiments only under favourable circumstances such as
colour-changes, accumulation techniques, etc. [ 9 ]. These processes, however, occur
in microscopy with high probability due to the (orders of magnitude) higher
excitation intensities and reduce the brightness [ 52 ]. Changes of t Fl in microscopy
were actually observed for CFPs, some photoactivatable proteins, dsRed and also
for enhanced GFP (eGFP) under the conditions of single-molecule spectroscopy
[ 53 - 56 ]. Early evidence was provided that the change of t Fl is due to photoconver-
sion (see Fig. 6 )[ 21 ]. Also, preferential bleaching of one conformer in CFP (see
below) with a longer lifetime was discussed [ 57 ], and one could also argue that
light-driven isomerization leads to a rotamer with reduced t Fl [ 58 , 59 ]. It is not clear
whether there is a general mechanism and, if so, whether it always leads to a
significant reduction of t Fl . A clear pattern, when a reduction of t Fl is likely to be
observed, is still missing.
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