Quantification of the Depolarization and Anisotropy of Fluorophore Stokes-Shifted Fluorescence, On-Resonance Fluorescence, and Rayleigh Scattering

Fluorophores are important but optically complicated photonic materials as they are simultaneous photon absorbers, emitters, and scatterers. Existing studies on fluorophore optical properties have been focused almost exclusively on its photon absorption and Stokes-shifted fluorescence (SSF) with scant information on the fluorophore photon scattering and on-resonance fluorescence (ORF). Presented herein is a unified theoretical framework and experimental approach for quantification of the fluorophore SSF, ORF, and scattering depolarization and anisotropy using a combination of fluorophore UV–vis, fluorescence emission, and resonance synchronous spectroscopic spectral measurements. A mathematical model for calculating fluorophore ORF and scattering cross sections has been developed that uses polystyrene nanoparticles as the external reference. The fluorophore scattering cross section is ∼10-fold smaller than its ORF counterparts for all the six model fluorophores, but more than 6 orders of magnitude larger than the water scattering cross section. Another finding is that the fluorophore ORF has a depolarization close to 1, while its Rayleigh scattering has zero depolarization. This enables the experimental separation of the fluorophore ORF and photon scattering features in the fluorophore resonance synchronous spectra. In addition to opening a new avenue for material characterization, the methods and insights derived from this study should be important for developing new analytical methods that exploit the fluorophore ORF and photon scattering properties.