Quantification of Material Fluorescence and Light Scattering Cross Sections Using Ratiometric Bandwidth-Varied Polarized Resonance Synchronous Spectroscopy

Abstract

Presented herein is the ratiometric bandwidth-varied polarized resonance synchronous spectroscopy (BVPRS2) method for quantification of material optical activity spectra. These include the sample light absorption and scattering cross-section spectrum, the scattering depolarization spectrum, and the fluorescence emission cross-section and depolarization spectrum in the wavelength region where the sample both absorbs and emits. This ratiometric BVPRS2 spectroscopic method is a self-contained technique capable of quantitatively decoupling material fluorescence and light scattering signal contribution to its ratiometric BVPRS2 spectra through the linear curve-fitting of the ratiometric BVPRS2 signal as a function of the wavelength bandwidth used in the PRS2 measurements. Example applications of this new spectroscopic method are demonstrated with materials that can be approximated as pure scatterers, simultaneous photon absorbers/emitters, simultaneous photon absorbers/scatterers, and finally simultaneous photon absorbers/scatterers/emitters. Because the only instruments needed for this ratiometric BVPRS2 technique are the conventional UV-vis spectrophotometer and spectrofluorometer, this work should open doors for routine decomposition of material UV-vis extinction spectrum into its absorption and scattering component spectra. The methodology and insights provided in this work should be of broad significance to all chemical research that involves photon/matter interactions.

Linear Extrapolation of the Analyte-Specific Light Scattering and Fluorescence Depolarization in Turbid Samples

“…16,17 For these samples, one must reliably separate material on-resonance fluorescence, the fluorescence emission at the same wavelength with the excitation photons, from the light scattering. 17…”

“…Although quantification of the material lightscattering depolarization spectrum for samples that are pure scatterers and simultaneous photon absorbers and scatterers is straightforward with this PRS2 method, caution should be exercised for fluorescent samples that are simultaneously photon absorbers, scatterers, and emitters. 16,17 For these samples, one must reliably separate material on-resonance fluorescence, the fluorescence emission at the same wavelength with the excitation photons, from the light scattering. 17 Although fluorescence and light scattering depolarization share the same mathematical forms, they differ significantly in origin, magnitude, and information content.…”

“…16,17 For these samples, one must reliably separate material on-resonance fluorescence, the fluorescence emission at the same wavelength with the excitation photons, from the light scattering. 17 Although fluorescence and light scattering depolarization share the same mathematical forms, they differ significantly in origin, magnitude, and information content. Fluorescence anisotropy is a function of the fluorophore fluorescence lifetime, angle between fluorophore excitation and emission dipoles, and rotational mobility in solutions.…”

Response on “commentary on “using resonance synchronous spectroscopy to characterize the reactivity and electrophilicity of biologically relevant sulfane sulfur”. Evidence that the methodology is inadequate because it only measures unspecific light scattering”. The evidence is incorrect

“…The produced S 8 should aggregate into fine particles similar to that of S 8 , obtained via diluting sulfur stock in acetone into the same buffer; both should display reduced RS 2 signals likely because of scattering and the sulfane sulfur property of S 8 (Fig. 1), as RS 2 is often used to analyzed aggregates of dye molecules [5,6].…”

“…RS 2 is a data acquisition method by using a fluorometer. RS 2 signals can be contributed by scattering, on-fluorescence, and possible Stokes’ shifted fluorescence [6]. Further, resonant Rayleigh scattering, caused by molecular polarity, may also contribute to RS 2 [7,8].…”