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Anal Biochem. 2011 Mar 1;410(1):62-9. doi: 10.1016/j.ab.2010.11.010. Epub 2010 Nov 13.

Applications of phasors to in vitro time-resolved fluorescence measurements.

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1
J. Heyrovský Institute of Physical Chemistry, v.v.i., Academy of Sciences of the Czech Republic, Dolejškova 3, Prague 18223, Czech Republic.

Abstract

The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented.

PMID:
21078290
PMCID:
PMC3065364
DOI:
10.1016/j.ab.2010.11.010
[Indexed for MEDLINE]
Free PMC Article
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