Application of Phasor Plots to Analysis of Fluorophore Heterogeneity, Excited State Reactions and Protein Conformations

Abstract

Phasor plots provide a simple graphical method to visualize and quantify time resolved fluorescence data, obtained using either frequency or time domain methods, independent of model constraints. Using the phase and modulation approach, the phasor plot converts raw data at a single frequency to a vector. Single exponential decays appear on the universal circle (semicircle with radius 0.5 and center 0.5, 0) whereas decays due to multiple exponentials appear as points inside the universal circle. This method has been successfully applied to fluorescence lifetime imaging microscopy (FLIM) wherein the data are typically collected at only one frequency. Applications of phasor plots in FLIM studies have, to date, been largely limited to FRET studies in cells. We have extended the application of phasor plots to several in vitro systems. Specifically, we have analyzed frequency-domain data of binary and tertiary mixtures of non-interacting, monoexponential-decay fluorophores and intrinsic protein fluorescence using the phasor plot approach. Phasor points from binary mixtures of varying composition lie along the line connecting the individual component points on the universal circle, while tertiary mixture points fall in a triangle between the individual vectors. Molecular interactions such as protein dissociation, protein-ligand interaction, denaturation, and energy transfer resulted in changes in the position of the vector point allowing for a rapid, graphical representation of these complex reactions. Data at a single frequency may be recorded rapidly allowing resolution of kinetic processes that would be difficult to monitor using complete multifrequency approaches. The combined results demonstrate the value of the phasor plot method to in vitro lifetime analysis. This work was supported in part by a grant from Allergan, Inc.