Fluorescence phasor analysis: basic principles and biophysical applications

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 nineteenth 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 parts 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 molecular 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.