Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 46

laser source, pump-induced changes in the sample optical density of 10 5 can be
observed. In transient difference measurements, the increased transmission (or
stimulated emission) appears as a negative signal (a 'bleach'), while induced
absorption appears as a positive signal.
In some measurements, both pump and probe wavelengths are the same and the
bleach and recovery of the ground state are observed. However, much more informa-
tion is available from measurements at a range of wavelengths spanning the induced
absorption and stimulated emission. To record such transient spectra, it is convenient
to probe with an ultrafast broadband source of radiation. For intense laser sources, such
broadband radiation can be obtained by white light continuum generation, in which an
intense pulse of 800 nm radiation (for example) is focused into a CaF 2 or sapphire
plate, which results in the generation of a short pulse of radiation with frequency
components stretching between the UV and near IR through a third-order non-linear
optical interaction [ 34 ]. This permits the simultaneous observation of the temporal
evolution of a range of transients. Quite recently, it has proved possible to generate
moderately broadband (approximately 500 cm 1 ) radiation in the mid-IR spectral
range. This permits the observation of photo-induced transients with both ultrafast
time resolution and vibrational frequency resolution [ 46 , 47 ]. Vibrational spectroscopy
is in general more informative on and more sensitive to the molecular structure. An
example showing the transient IR difference spectra of the HBDI chromophore is
shown in Fig. 3 . For either visible or IR probes, the entire 3D time-frequency-intensity
surface can be simultaneously analysed by global analysis methods [ 48 ].

3 Photophysics in HBDI

Given its name, one initially surprising feature of the GFP chromophore (HBDI) is that it
does not in fact fluoresce, or at least not very much; in room temperature aqueous
solution, the quantum yield is on the order of 10 4 . This low quantum yield is also
observed in the denatured protein and short lengths of peptide containing the chromo-
phore [ 13 ]. These results are in sharp contrast to avGFP, where the quantum yield for
emission is about 0.8 [ 6 ]; evidently the folded protein structure has a profound effect on
the radiationless decay of the chromophore. An important challenge in GFP photophy-
sics is to understand both the mechanism of the extremely rapid radiationless decay and
the means by which the protein matrix can suppresses it so efficiently. Significantly a
number of the more recently discovered CPs which contain the same basic chromophore
as avGFP, and share essentially the same -barrel structure, are non-fluorescent or only

very weakly fluorescent [ 15 , 49 ]. Thus, an understanding of radiationless decay in HBDI
is central to understanding the mechanism of operation of photoswitchable CPs.

b

3.1 HBDI Spectroscopy

In Fig. 4 , the absorption spectra of HBDI in the neutral and anionic states are
compared with that of avGFP, which exists mainly as the neutral form but with