Effects of Cascading Optical Processes: Part III. Impacts on Spectroscopic Measurements of Fluorescent Samples

Abstract

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.

Advancing Evidence-Based Data Interpretation in UV–Vis and Fluorescence Analysis for Nanomaterials: An Analytical Chemistry Perspective

“…The analytical chemistry model (eq ) is far more complex than Beer’s law. eq is derived by parametrizing the experimental UV–vis extinction E UV (λ x ) as the analyte extinction, scattering extinction, and sampling conditions . For generality, the analytes are assumed to be simultaneous absorbers, scatterers, and emitters under excitation wavelength λ x . …”

“…Experimental confirmation of the scattering and fluorescence interference on UV–vis measurements is straightforward by studying the sample UV–vis LDR and by comparing the conventional UV–vis spectrum with that acquired with the integrating-sphere-assisted UV–vis method (ISUV). − For molecular chromophores that are approximately pure absorbers, the experimental UV–vis spectrum maintains excellent linearity if the sample optical density is within the instrument LDR specified by the vendor (Figure S1­(A–D)). However, for nanoparticles that are scatterers, simultaneous absorbers and scatterers, or simultaneous absorbers, scatterers, and emitters, the upper limit of the experimental LDR can be significantly lower than that for the pure absorbers (Figure S1­(E–L)).…”

“…Although scattering itself does not produce fluorescence, as rightfully implied by eq , it can change the locations and number of photons absorbed as well as the path of emitted photons (Figure B). Compared to the light absorption, however, the impact of scattering on fluorescence is significantly smaller. , Empirically, the effects of light scattering on the fluorophore fluorescence can be modeled by adding two additional terms into eq , depicting two competing effects of scattering on fluorophore fluorescence. The η­( S ) term in eq represents the scattering inner filter effect, depicting the extent of fluorescence signal reduction caused by the scattering on the excitation photons propagating to the effective sampling path length defined by the instrument pinhole effects (Figure B).…”

“…Unlike light absorption, which terminates the excitation or emission photons, scattering changes only the propagation paths of the excitation photons. These scattered photons remain effective for fluorescence generation before exiting the cuvette, and some of these emitted photons can propagate to the fluorescence detector, partially compensating for the scattering inner filter effect on fluorescence signal reduction …”

“…Another important step in mitigating ambiguous UV–vis data analysis is to report the as-acquired UV–vis spectra with clearly labeled spectral intensities, rather than solely relying on normalized UV–vis data or simply ignore the scale of the UV–vis intensity. In nano science literature, it has been quite common to present normalized or arbitrarily scaled and/or baseline-offset spectra obtained from materials of varying sizes, concentrations, and compositions in the same figure. − While these approaches might be effective to highlight spectral differences among the samples, it can obscure potential issues in data reliability as UV–vis spectra can be highly distorted even when the sample UV–vis intensity is within the instrument LDR. − To promote evidence-based spectral interpretation, we believe it is imperative to avoid reporting the processed UV–vis spectra without the as-acquired spectra either in the Supporting Information or in the main text.…”