the cross-section of the phenolate species is twice that of the neutral species and taking
into account the lower fluorescence quantumyield of the anionic species relative to the
neutral state. The ratio between the photobleaching and photoconversion rate con-
stants is pH dependent and showed a minimum at pH 8.0, from comparing the
photogenerated absorption at 483 nm relative to ground state absorption decrease at
398 nm [ 19 ]. Under these conditions, a close to stoichiometric photoconversion of the
neutral species to the anionic GFP R species could be achieved with UV illumination at
254 nm [ 19 ].
Cubitt et al. already noted that the photoconversion can be used to monitor
diffusion or migration of GFP fusion proteins after spatially localised photo-
isomerisation [ 7 ]. Indeed, such approaches have been taken very successfully
[ 33 - 35 ].
The kinetics of photoconversion were investigated between 102K and 293K, but
very little temperature dependence could be observed. Assuming Arrhenius beha-
viour, an activation energy of about 1 KJ/mol could be estimated [ 19 ]. However,
performing photoconversion at cryogenic temperatures revealed thermal relaxation
processes that are characterised by blue-shifted electronic absorption of the anionic
photoproduct state. These relaxation processes were studied by time-resolved cryo
X-ray crystallography and cryo-FTIR spectroscopy [ 26 ].
Both UV and visible (blue and green) light lead to photoconversion of GFP.
However, photobleaching is significant under conditions of pulsed blue excitation.
Figure 6 compares the absorption changes under visible (390 nm) and UV (254 nm)
excitation. Kinetic analysis of the macroscopic photoconversion rate as a function
of the power density of the nanosecond pulsed laser at 390 nm indicated a linear
relationship with regard to the photoconversion rate [ 19 , 36 ]. This shows that under
the optical regime that was used, the reaction proceeds via a one photon process.
Photoconversion also proceeds with intense continuous illumination at 476 nm
[ 37 ], or femtosecond pulsed excitation at 478 nm [ 16 ], directly exciting the minor
anionic GFP B species. The quantum yield appears to be approximately 100 times
below that with 254 nm excitation [ 37 ]. Two- or multiphoton photoconversion
using 800-nm pulses was also reported [ 38 ].
An interesting and currently unresolved issue regarding blue light-induced
photoconversion is that its rate constant may not depend linearly on the power
density when continuous illumination is used, in contrast to nanosecond pulsed
excitation. For this particular regime, the evidence available in the literature is less
complete, and only single observations are reported. For example, Patterson and
Lippincott-Schwarz observed second timescale photoconversion rates with 413 nm
continuous excitation from an ion laser [ 35 ]. The power used was reported (1 mW),
but not the spot size. When alternatively a 100 W Hg + lamp together with a 405-nm
bandpass filter was used, photoconversion proceeded in ~60 s in their microscope
setup [ 35 ]. Again, no power density was reported, but the power density effect of
continuous illumination is clear. However, weaker blue illumination at 404 nm and
6 mW/cm 2 has been reported to be ineffective [ 37 ]. In the absence of a systematic
investigation on the power dependence over these different regimes, no conclusion
is presently possible. Clearly, two photon photoconversion with CW illumination at