Applications of Phasor Plots to Dynamic Polarization Studies

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 Forster resonance energy transfer. Phasors can be calculated using either frequency domain or time-domain data. The phasor approach has become a valuable tool for both in vitro cuvette studies and in fluorescence lifetime imaging microscopy (FLIM) studies with live cells. In addition to intensity decay, e.g., lifetime data, time-resolved fluorescence can be used to provide information on the rotational mobilites of molecules. This information is acquired in the time-domain using time-decay anisotropy or its frequency domain equivalent known as dynamic polarization. Here, we apply the phasor method to frequency domain dynamic polarization data to provide graphical information on the rotational modalities of fluorophores in vitro. The method is illustrated using a series of simple model systems including fluorophores in isotropic solvents of varying viscosities as well as more complex systems including ethidium bromide interaction with tRNA and also proteins exhibiting varying extents of tryptophan motion. This work was supported in part by funding from Allergan, Inc.