Fluorescence Phasor Analysis: Basic Principles and Biophysical Applications

TL;DR

This paper reviews the principles of fluorescence phasor analysis, a powerful, model-free technique for analyzing complex fluorescence data in biophysics, with applications in studying protein dynamics, lipid phase transitions, and nucleic acid structures.

Contribution

It provides a comprehensive overview of fluorescence phasor analysis, highlighting its fundamental properties and diverse biophysical applications, emphasizing its intuitive and model-free nature.

Findings

Phasor analysis enables intuitive interpretation of fluorescence data.

Application to protein folding and interactions demonstrates its versatility.

Effective in studying lipid phase transitions and nucleic acid structures.

Abstract

Fluorescence is one of the most widely used techniques in biological sciences. Its exceptional sensitivity and versatility make it a tool of first choice for quantitative studies in biophysics. The concept of phasors, originally introduced by Charles Steinmetz in the late 19th century for analyzing alternating current circuits, has since found applications across diverse disciplines, including fluorescence spectroscopy. The main idea behind fluorescence phasors was posited by Gregorio Weber in 1981. By analyzing the complementary nature of pulse and phase fluorometry data, he shows that two magnitudes -- denoted as $G$ and $S$ -- derived from the frequency-domain fluorescence measurements correspond to the real and imaginary part of the Fourier transform of the fluorescence intensity in the time domain. This review provides a historical perspective on how the concept of phasors originates and how it integrates into fluorescence spectroscopy. We discuss their fundamental algebraic properties, which enable intuitive model-free analysis of fluorescence data despite the complexity of the underlying phenomena. Some applications in biophysics illustrate the power of this approach in studying diverse phenomena, including protein folding, protein interactions, phase transitions in lipid mixtures and formation of high-order structures in nucleic acids.