Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 88

Experimental data provide evidence for this mechanism since only in this case
changes in the vicinity of the imidazolinone heterocycle, e.g. by photoconversion
should influence t Fl [ 21 ]. According to recent theoretical results, it seems that
enough spatial freedom is given for efficient radiationless processes even in YFPs
[ 69 ]. This finding could explain why t Fl of many YFPs, despite the steric constraints
of Y203, is not superior to eGFP, i.e., around 3.0 ns [ 48 ]. One should mention at this
point that the most widely used YFPs contain the mutation S65G for enhanced
maturation, which is shown to distinctly reduce F Fl as well as t Fl [ 21 ].

3.3.3 Some Peculiarities

Although the preceding paragraphs might evoke the impression that especially the
photophysics of anionic chromophore species is completely understood, some
astonishing facts should be listed, which deserve further investigations:

1. Red-edge excitation of wt-GFP yielded t Fl ¼ 4.5 ns which is, obviously, close

to t rad [ 79 ]. There is hope that even with GFPs, higher t Fl like for CFPs can be
achieved.
2. Even considerably longer t Fl of 6.2 ns (averaged value) of green emitting forms
of dsRed were observed in single-molecule experiments [ 89 ]. This value is the
longest t Fl ever measured for GFP-like molecules, and immediately raises the
question whether the green emitting form of dsRed is different from avFP
in terms of its electronic properties. Another issue is whether energy migration,
i.e., homo-FRET [ 20 ], can explain such a strong prolongation as it is observed
for ordinary reabsorption [ 90 ]. Many FPs form oligomers (dsRed is only a
prominent example), so this process may have practical relevance when apply-
ing these FPs in cell imaging applications.
3. The preceding example highlights the analytical power of single-molecule experi-
ments. Such investigations are also successful in showing that intense illumination
leads to a reduction of t Fl of eGFP [ 54 ], whereas ensemble experiments on the
very similar S65T failed (see Fig. 6 ). Single-molecule experiments are beneficial
when the disappearance of some intermediate is faster than its generation: under
these kinetic conditions, a small population with atypical behaviour can be easily
overseen in ensemble investigations. It is still the question why some lifetime
measurements in cellular environments and microscopy yield t Fl , which are

considerably smaller than in cuvette experiments [ 47 , 50 ].

3.4 Orange and Red Fluorescent Proteins

With the examination of more animals, from which FPs could be isolated after-
wards, FPs with emission maxima l >

530 nm became available [ 91 , 92 ]. At the
beginning, especially dsRed is investigated and the basis for further developments.