Fluorophore maturation

Ratio imaging is particularly suited for single-polypeptide FRET sensors. In these constructs FRET changes are due to altered distance and/or orientation of the donor and acceptor, and since the fluorophores are tethered their stoichiometry is always fixed. Thus, the filterFRET problems are easier to address and, assuming full maturation of both FPs [4], it can in fact be shown that under these circumstances two images suffice to calculate FRET quantitatively (see Textbox 1 and Appendix 7.A.6). 

Many of the strategies for measuring FRET from spectral images that were mentioned above have been implemented to study FRET. We will now cover sRET [12], a specific implementation that uses the last approach where FRET is measured from a pair of spectral images collected at different excitation wavelengths. Recently, the sRET approach has been extended to explicitly consider paired and unpaired fluorophores, the impact of incomplete labeling (or for fluorescent proteins fractional maturation), and the... 

Fluorescence polarization is a dimensionless ratio with values from 0.000 to 0.500 for dilute solutions containing fluorescing compounds (see Chapter 3). Polarization (P) measures the rotational diffusion of the fluorophore relative to its fluorescent half-life. If the half-life is short compared with the rate of rotational diffusion, P will be high. In contrast, if molecular rotation is faster than the excited state decay, then P wfll be low. Shinitzky proposed that for amni-otic fluid, lower polarization values reflected a decrease in the microviscosity of surfactant phospholipids. The fluidity of these phospholipids paralleled the change in lipid composition with maturation of the fetal lungs. This mechanism is incorrect for the NBD-PC method. NBD-PC binds to albumin and to surfactant thus the resulting polarization is a function of the surfactant/albumin ratio. ... 

As a further drawback, native DsRed also exhibits slow and complex fluo-rophore maturation due to an additional autocatalytic modification, extending the chromophore s conjugation system and allowing for red fluorescence [44, 45]. Non-mature protein with 475-nm excitation/500-nm emission maxima transforms into mature protein with 558-nm excitation/585-nm emission maxima and requires >48 h to reach 90% of maximal fluorescence [39]. Fluo-rophore maturation has been significantly accelerated in a nimiber of mutants, e.g. Tl, mRFPl and E57 and was proposed to depend upon the space arovmd the fluorophore [46,47]. 

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