Fluorescence switching behaviour

Figure 32 Thermodynamic bases of the fluorescence switching behaviour illustrated in Figure 30. The eT process from the Ni " centre to the nearby photo-excited dansyl subunit (dns ) is characterized by a strongly negative free energy change ACj°eT = —1-93 eW, as calculated through the combination of pertinent photophysical and el ti -chemical quantities. On the other hand, the dns -to-Ni eT process is very disfavoured (AG°eT>0.68 eV).

Dickson R M, Cubitt A B, Tsien R Y and Moerner W E 1997 On/off blinking and switching behaviour of single molecules of green fluorescent protein Nature 388 355-8... 

R. M. Dickson, A.B. Cubitt, R.Y Tsien, and W. E. Moerner, On/Off Blinking and Switching Behaviour of Single Molecules of Green Fluorescent Protein, Nature 388,... 

There is an interesting structural similarity between (66) and the other subjects of this section -(67) [149], (68) [150], (69) [151] and (70) [152,153] - in that they are all composed of 4-aminophenyl groups substituted at a meso position of a linearly fused tricyclic aromatic frame. The similarity continues even in their experimental behaviour in that protonation causes significant fluorescence enhancements in all cases. The structural elaboration of the aniline unit in (70) allows similar switching on with calcium ions. The case (70) is taken up further in Sect. 7. Additionally, (69) with its very electron deficient acridinium acceptor unit shows extensive perturbation of absorption spectra by several cations. Again, the acidity constants are experimentally identical in both the ground and excited singlet states. We close this final section of the survey... 

Gate 42 [97] is interesting because various transition metal ions serve to switch on fluorescence by more-or-less same amounts. The surprise in this system [97, 98] is that transition metal ions were acting contrary to their normal behaviour. Transition metal ions have a history of quenching fluorescence very efficiently by several mechanisms heavy atom effects, PET, electronic energy transfers and... 

A third potential threat to proper FRAP analysis is the blinking behaviour of GFP. It has been shown that many fluorescent proteins rapidly switch between a dark non-fluorescent state and a fluorescent state. The period GFPs are in the dark state (off-time) is not dependent on laser whereas the on-times are [37,38]. Since the bleach pulse is at a much higher intensity as the monitoring after bleaching, it can be expected that a part of the recovery of fluorescence is due to decrease of the pool of molecules in the off-state. This is not a great problem if all conditions are kept constant, since it will only introduce a constant bias in the results However, comparison between different variants of GFP that have different blinking properties is hampered. 

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