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Fig. 1 Jablonski scheme for chromophores. With respect to fluorescent proteins, most spectro-
scopic data exist for absorption, emission and internal conversion ( black arrows ), i.e., transitions
between the lowest two singlet-states S 0 and S 1 . Experimental findings hint at some relevance of
higher excited singlet-states S n and the lowest triplet state T 1 ( grey, full line arrows ). Little is
known about other possible transitions ( grey, dotted line arrows )
of aromatic amino acids also absorbs strongly in the UV-range. Selective destruc-
tion of the chromophores in FPs, which would allow for verifying the energetic
position in the natural environment by subtracting the spectra after some chemical
treatment from the original spectra, is possible but likely not reliable enough [ 5 ].
More reliable is experimental and theoretical work on two-photon excitation, which
gives insight into the energetics of higher excited states [ 6 , 7 ]. That these higher
excited singlet states are indeed of more than academic interest, can be inferred
from two-photon excitation microscopy [ 8 ] and from the enhanced photoconver-
sion upon excitation in these states [ 9 , 10 ]: it was shown that both UV-excitation at
254 nm and excited-state absorption of the neutral chromophore lead to a rapid
decarboxylation of a nearby glutamate at position 222. Based on these experiments,
one could also hypothesize that other photochemical reactions such as photobleach-
ing are accelerated upon excitation into higher excited states. Especially the
characterization of excited-state absorption would be desirable since pump-dump
schemes such as in stimulated-emission depletion (STED)-microscopy might suffer
from such detrimental reactions [ 11 , 12 ]. Excited-state absorption might also occur
to some extent in microscopy within the diffraction-limited spot of a focussed laser
beam, even with continuous-wave excitation. Due to the yet limited knowledge
about these processes, we restrict the following discussion to the lowest excited
states, i.e., S 1 and T 1 .
The relevance of the lowest energetic triplet state is still under debate. In the
past, fluorescence correlation spectroscopy (FCS) gave some evidence that the
triplet state is significantly populated at high intensities in FPs [ 13 ]. Unfortunately,
FCS only detects transient states due to their missing fluorescence emission, which
is especially ambiguous in FPs: there are more photochemical reactions, which can
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