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.

The sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometric-based measurements treat light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling of the photon absorption and scattering contribution. The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometer equipped with a pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.

Existing studies on molecular fluorescence have almost exclusively been focused on Stokes-shifted fluorescence spectroscopy (SSF) in which the emitted photon is detected at the wavelengths longer than that for the excitation photons. Information on fluorophore on-resonance fluorescence (ORF) and resonance Rayleigh scattering (RRS) is limited and often problematic due to the complex interplay of the fluorophore photon absorption, ORF emission, RRS, and solvent Rayleigh scattering. Reported herein is a relatively large-scale systematic study on fluorophore ORF and RRS using the conventional UV-vis extinction and SSF measurements in combination with the recently reported ratiometric resonance synchronous spectroscopic (R2S2, pronounced as "R-Two-S-Two") method. A series of fundamental parameters including fluorophore ORF cross sections and quantum yields have been quantified for the first time for a total of 12 molecular and 6 semiconductor quantum dot (QD) fluorophores. All fluorophore spectra comprise a well-defined Gaussian peak with a full width at half-maximum ranging from 4 to 30 nm. However, the RRS features of fluorophores differ drastically. The effect of fluorophore aggregation on its RRS, UV-vis, R2S2, and SSF spectra was also discussed. This work highlights the critical importance of the combined UV-vis extinction, SSF, and R2S2 spectroscopic measurements for material characterizations. The method and insights described in this work can be directly used for improving the reliability of RRS spectroscopic methods in chemical analysis. In addition, it should pave the way for developing novel R2S2-based analytical applications.

With their unique photochemical properties, porphyrins have remained for decades the most interested chemicals as photonic materials for applications ranging from chemistry, biology, medicine, to photovoltaic. Porphyrins can self-assemble into higher order structures. However, information has been scant on the kinetics and structural evolution during porphyrin assembly and disassembly. Furthermore, quantitative understanding of the porphyrin optical activities is complicated by the complex interplay of photon absorption, scattering, and fluorescence emission that can concurrently occur in porphyrin samples. Using meso-tetrakis(4-sulfonatophenyl)porphyrin as the model molecule, reported herein is a combined UV-vis extinction, polarized Stokes-shifted fluorescence, and polarized resonance synchronous spectroscopic (PRS2) study of porphyrin assembly and disassembly in acidic solutions. Although porphyrin assembly and disassembly occur instantaneously upon the sample preparation, both processes last at least a few months before reaching their approximate equilibrium states. The two processes were monitored in situ by quantifying the porphyrin fluorescence and scattering depolarizations as well as its extinction, absorption, scattering, and fluorescence emission cross sections. In addition to a series of new insights to the porphyrin assembly and disassembly, the methodology described in this work opens the door for the in situ study of the structural and optical properties of photonic materials comprising molecular assembly.