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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. 

Cascading optical processes involve sequential photon–matter interactions triggered by the same individual excitation photons. Parts I and II of this series explored cascading optical processes in scattering-only solutions (Part I) and solutions with light scatterers and absorbers but no emitters (Part II). The current work (Part III) focuses on the effects of cascading optical processes on spectroscopic measurements of fluorescent samples. Four types of samples were examined: (1) eosin Y (EOY), an absorber and emitter; (2) EOY mixed with plain polystyrene nanoparticles (PSNPs), which are pure scatterers; (3) EOY mixed with dyed PSNPs, which scatter and absorb light but do not emit; and (4) fluorescent PSNPs that are simultaneous light absorbers, scatterers, and emitters. Interference from both forward scattered and emitted photons can cause nonlinearity and spectral distortion in UV–vis extinction measurements. Sample absorption by nonfluorogenic chromophores reduces fluorescence intensity, while the effect of scattering on fluorophore fluorescence is complicated by several competing factors. A revised first-principles model is developed for correlating the experimental fluorescence intensity with the sample absorbance in solutions containing both scatterers and absorbers. The optical properties of fluorescent PSNPs of three different sizes were systematically investigated by using integrating-sphere-assisted resonance synchronous spectroscopy, linearly polarized resonance synchronous spectroscopy, UV–vis, and fluorescence spectroscopy. The insights and methodology provided in this work should help improve the reliability of spectroscopic analyses of fluorescent samples, where the interplay among light absorption, scattering, and emission can be complex.