On-Resonance Fluorescence, Resonance Rayleigh Scattering, and Ratiometric Resonance Synchronous Spectroscopy of Molecular- and Quantum Dot-Fluorophores

Abstract

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.

Determining the Liquid Light Scattering Cross Section and Depolarization Spectra Using Polarized Resonance Synchronous Spectroscopy

“…The lacking of ORF interference is due most likely to the combined effect of the low fluorescence impurity concentration and the low ORF quantum yield. Earlier study showed that the ORF quantum yield for a molecular fluorophore is usually smaller than ∼0.01 …”

“…Earlier study showed that the ORF quantum yield for a molecular fluorophore is usually smaller than ∼0.01. 20 Filtration has been used extensively for removing particulates in light scattering measurements. 14,18,35,36 However, care must be exercised in using filtration.…”

“…This is because one must subtract the solvent light scattering contribution in the determination of the analyte light scattering and possible on-resonance fluorescence (ORF) cross sections. 19,20 ORF is the fluorescence photon emission that occurs in the same wavelength as the excitation wavelength. Earlier light scattering work often mistakes ORF as light scattering.…”

“…Filling this knowledge gap for liquid samples is important for studying the liquid structure and properties correlations and also for reliable experimental quantification of the optical properties of analytes dissolved in solvents. This is because one must subtract the solvent light scattering contribution in the determination of the analyte light scattering and possible on-resonance fluorescence (ORF) cross sections. , ORF is the fluorescence photon emission that occurs in the same wavelength as the excitation wavelength. Earlier light scattering work often mistakes ORF as light scattering. − …”

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

“…16 With this method, we have quantified for the first time the ORF emission cross sections and quantum yields for a series of molecular and quantum dot fluorophores. 17 While the R2S2 spectroscopic technique provides a convenient way for experimental correction of the sample inner filter effect (IFE) imposed by the fluorophore photon absorption on the combined fluorophore photon scattering and ORF contribution to experimental resonance synchronous spectrum (RS2), separation of the fluorophore ORF and photon scattering contribution to the IFE-corrected R2S2 spectrum is, however, challenging. It was performed through a spectral subtraction method on theoretical predication that the peak shape of the ORF is equivalent to the multiplication spectrum of the fluorophore UV−vis and SSF spectra.…”