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Fig. 6 Intensity-dependent change of t Fl due to diffraction-limited focusing of a pulsed laser
(l exc ΒΌ 470 nm, 63 NA 1.4 objective lens). The FPs were freely diffusing in a buffered solution
at micromolar concentrations. This phenomenon was described for CFP [ 55 , 57 ], dsRed [Bowen
and Woodbury 2003] and even for eGFP [ 54 ]. Some GFP mutants such as T203V (see [ 56 ]) and
C48S exhibit a change of t Fl , whereas the widely used mutants T203H and S65T appear stable in
t Fl . The reason for this finding is unclear: preferential bleaching of one conformer, photoconver-
sion or the light-induced population of some metastable isomer are discussed
2.4 Search for Proteins with Improved Photophysical Properties
As will be shown in Sect. 3 , t Fl depends on the mutation pattern, and the under-
standing of the decay mechanisms allows for designing FPs with altered t Fl . Despite
this rational proceeding, the progress is limited as protein folding, chromophore
maturation and other biochemical parameters are also influenced by mutations.
A rather elegant and straightforward approach unifies the rapid amplification of
DNA by polymerase-chain reaction (PCR), the rapid conversion of DNA into
proteins by bacteria and, finally, the screening capabilities of fluorescence lifetime
imaging microscopy (FLIM) [ 48 , 49 , 60 ]. Accidental or specific mismatches during
PCR actually create a multitude of sequences, which can subsequently result in a
huge library of FPs with altered photophysical properties. On the one hand, any
disturbance of the chromophore formation immediately will abort fluorescence. On
the other hand, since t Fl can be measured independent from the fluorescence
intensity, FP mutants with prolonged t Fl can be found as long as some visible
protein fluorescence is detected. The expression yield can be optimized afterwards.
The success of this approach, however, strongly relies on the way how the DNA
library is generated. So far, most random mutagenesis only lead to a reduction of
t Fl , whereas restriction of the combinatorial approach to few important positions
enabled, quite recently, the generation of CFPs with F Fl >
90% and long t Fl (~4 ns)
near t rad [ 61 ].
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