Insights into Spectral Distortion and Nonlinearity in UV-Vis and Fluorescence Spectroscopy of Molecular Fluorophore...

Reproducibility and linearity are crucial benchmarks for any measurement technology. However, UV-vis and fluorescence spectral distortion and nonlinearity are prevalent, even in seemingly simple fluorescent solutions that comprise only one dissolved molecular fluorophore, without exogenous absorbing or scattering species. In this report, we introduce an analytical model for the quantification of fluorescence interference on UV-vis measurements and a conceptual model for mechanistically understanding the impacts of higher-order cascading optical processes on fluorescence measurements. The experimental UV-vis transmittance can be dominated by interfering fluorescence, even for fluorophore solutions with theoretical absorbance values far below the instrument's linear dynamic range (LDR). Absorption-inner-filter-effect (aIFE) correction drastically improves the fluorescence LDR. However, the efficacy of aIFE correction hinges on two competing factors that strongly depend on the fluorophore's optical properties: the degree of fluorescence interference in UV-vis and the significance of secondary or higher-order emission triggered by fluorophore absorption of emitted photons. Our research sheds light on the remarkable complexity of cascading optical processes that can occur even in the simplest fluorescent solutions. It emphasizes the necessity of critically evaluating optical spectroscopic measurements of fluorescent solutions to improve the reliability of analyzing and interpreting optical spectra. Moreover, it lays the groundwork for future development of methods capable of handling challenging samples that exceed the capabilities of the current tools.