Insights into Spectral Distortion and Nonlinearity in UV-Vis and Fluorescence Spectroscopy of Molecular Fluorophore Solutions: Effects of Cascading Optical Processes (Part IV).

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.

Multiplexed Surface Protein Detection and Cancer Classification Using Gap-Enhanced Magnetic-Plasmonic Core-Shell Raman Nanotags and Machine Learning Algorithm.

Cancer is the second leading cause of death attributed to disease worldwide. Current standard detection methods often rely on a single cancer marker, which can lead to inaccurate results, including false negatives, and an inability to detect multiple cancers simultaneously. Here, we developed a multiplex method that can effectively detect and classify surface proteins associated with three distinct types of breast cancer by utilizing gap-enhanced Raman scattering nanotags and machine learning algorithm. We synthesized anisotropic magnetic core-gold shell gap-enhanced Raman nanotags incorporating three different Raman reporters. These multicolor Raman nanotags were employed to distinguish specific surface protein markers in breast cancer cells. The acquired signals were deconvoluted and analyzed using classical least-squares regression to generate a surface protein profile and characterize the breast cancer cells. Furthermore, computational data obtained via finite-difference time-domain and discrete dipole approximation showed the amplification of the electric fields within the gap region due to plasmonic coupling between the two gold layers. Finally, a random forest classifier achieved an impressive classification and profiling accuracy of 93.9%, enabling effective distinguishing between the three different types of breast cancer cell lines in a mixed solution. With the combination of immunomagnetic multiplex target specificity and separation, gap-enhancement Raman nanotags, and machine learning, our method provides an accurate and integrated platform to profile and classify different cancer cells, giving implications for identification of the origin of circulating tumor cells in the blood system.

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

UV-vis spectrophotometry and spectrofluorometry are indispensable tools in education, research, and industrial process controls with widespread applications in nanoscience encompassing diverse nanomaterials and fields. Nevertheless, the prevailing spectroscopic interpretations and analyses often exhibit ambiguity and errors, particularly evident in the nanoscience literature. This analytical chemistry Perspective focuses on fostering evidence-based data interpretation in experimental studies of materials' UV-vis absorption, scattering, and fluorescence properties. We begin by outlining common issues observed in UV-vis and fluorescence analysis. Subsequently, we provide a summary of recent advances in commercial UV-vis spectrophotometric and spectrofluorometric instruments, emphasizing their potential to enhance scientific rigor in UV-vis and fluorescence analysis. Furthermore, we propose potential avenues for future developments in spectroscopic instrumentation and measurement strategies, aiming to further augment the utility of optical spectroscopy in nano research for samples where optical complexity surpasses existing tools. Through a targeted focus on the critical issues related to UV-vis and fluorescence properties of nanomaterials, this Perspective can serve as a valuable resource for researchers, educators, and practitioners.

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

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.

Total Luminescence Spectroscopy for Quantification of Temperature Effects on Photophysical Properties of Photoluminescent Materials.

Quantification of the temperature effects on the optical properties of photoluminescent (PL) materials is important for a fundamental understanding of both materials optical processes and rational PL materials design and applications. However, existing techniques for studying the temperature effects are limited in their information content. Reported herein is a temperature-dependent total photoluminescence (TPL) spectroscopy technique for probing the temperature dependence of materials optical properties. When used in combination with UV-vis measurements, this TPL method enables experimental quantification of temperature effects on fluorophore fluorescence intensity and quantum yield at any combination of excitation and detection wavelengths, including the fluorophore Stokes-shifted and anti-Stokes-shifted fluorescence. All model polyaromatic hydrocarbon (PAH) and xanthene fluorophores exhibited a strong excitation- and emission-wavelength dependence in their temperature effects. However, the heavy-atom effects used for explaining the strong temperature dependence of brominated anthracenes are not operative with xanthene fluorophores that have heavy atom substitutions. The insights from TPL measurements are important not only for enhancing the fundamental understandings of the materials photophysical properties but also for rational measurement design for applications where the temperature sensitivity of the fluorophore fluorescence is critical. An example application is demonstrated for developing a sensitive and robust ratiometric fluorescence thermometric method for in situ real-time monitoring of sample temperatures inside a fluorescence cuvette placed in a temperature-controlled sample holder.

Effects of Cascading Optical Processes: Part II: Impacts on Experimental Quantification of Sample Absorption and Scattering Properties.

In Part I of the three companion articles, we reported the effects of light scattering on experimental quantification of scattering extinction, intensity, and depolarization in solutions that contain only scatterers with no significant absorption and photoluminescence activities. The present work (Part II) studies the effects of light scattering and absorption on a series of optical spectroscopic measurements done on samples that contain both absorbers and scatterers, but not emitters. The experimental UV-vis spectrum is the sum of the sample absorption and scattering extinction spectra. However, the upper limit of the experimental Beer's-law-abiding extinction can be limited prematurely by the interference of forward scattered light. Light absorption reduces not only the sample scattering intensity but also the scattering depolarization. The impact of scattering on sample light absorption is complicated, depending on whether the absorption of scattered light is taken into consideration. Scattering reduces light absorption along the optical path length from the excitation source to the UV-vis detector. However, the absorption of the scattered light can be adequate to compensate the reduced light absorption along such optical path, making the impacts of light scattering on the sample total light absorption negligibly small (<10%). The latter finding constitutes a critical validation of the integrating-sphere-assisted resonance synchronous spectroscopic method for experimental quantification of absorption and scattering contribution to the sample UV-vis extinction spectra. The techniques and general guidelines provided in this work should help improve the reliability of optical spectroscopic characterization of nanoscale or larger materials, many of which are simultaneous absorbers and scatterers. The insights from this work are foundational for Part III of this series of work, which is on the cascading optical processes on spectroscopic measurements of fluorescent samples.

Effects of Cascading Optical Processes: Part I: Impacts on Quantification of Sample Scattering Extinction, Intensity, and Depolarization.

Light scattering is a universal matter property that is especially prominent in nanoscale or larger materials. However, the effects of scattering-based cascading optical processes on experimental quantification of sample absorption, scattering, and emission intensities, as well as scattering and emission depolarization, have not been adequately addressed. Using a series of polystyrene nanoparticles (PSNPs) of different sizes as model analytes, we present a computational and experimental study on the effects of cascading light scattering on experimental quantification of NP scattering activities (scattering cross-section or molar coefficient), intensity, and depolarization. Part II and Part III of this series of companion articles explore the effects of cascading optical processes on sample absorption and fluorescence measurements, respectively. A general theoretical model is developed on how forward scattered light complicates the general applicability of Beer's law to the experimental UV-vis spectrum of scattering samples. The correlation between the scattering intensity and PSNP concentration is highly complicated with no robust linearity even when the scatterers' concentration is very low. Such complexity arises from the combination of concentration-dependence of light scattering depolarization and the scattering inner filter effects (IFEs). Scattering depolarization increases with the PSNP scattering extinction (thereby, its concentration) but can never reach unity (isotropic) due to the polarization dependence of the scattering IFE. The insights from this study are important for understanding the strengths and limitations of various scattering-based techniques for material characterization including nanoparticle quantification. They are also foundational for quantitative mechanistic understanding on the effects of light scattering on sample absorption and fluorescence measurements.

Integrating-Sphere-Assisted Resonance Synchronous Spectroscopy for the Quantification of Material Double-Beam UV-Vis Absorption and Scattering Extinction.

Integrating spheres (IS) have been used extensively for the characterization of light absorption in turbid samples. However, converting the IS-based sample absorption coefficient to the UV-vis absorbance quantified with a double-beam UV-vis spectrophotometer is challenging. Herein, we report an integrating-sphere-assisted resonance synchronous (ISARS) spectroscopy method performed with conventional spectrofluorometers equipped with an integrating-sphere accessory. Mathematical models and experimental procedures for quantifying the sample, solvent, and instrument-baseline ISARS intensity spectra were provided. A three-parameter analytical model has been developed for correlating the ISARS-based UV-vis absorbance and the absorbance measured with double-beam instruments. This ISARS method enables the quantitative separation of light absorption and scattering contribution to the sample UV-vis extinction spectrum measured with double-beam UV-vis spectrophotometers. Example applications of this ISARS technique are demonstrated with a series of representative samples differing significantly in their optical complexities, from approximately pure absorbers, pure scatterers, to simultaneous light absorbers, scatterers, and emitters under resonance excitation and detection conditions.

Back to the Drawing Board: A Unifying First-Principle Model for Correlating Sample UV-Vis Absorption and Fluorescence Emission.

The popular textbook and literature model I(λx,λm) = K(λx,λm)(1-10-Ax) or its variants for correlating the sample absorption and fluorescence often fails even for the simplest samples where the fluorophore is the only light absorber. Reported is a first-principle model I(λx,λm) = K(λx,λm)Ax,f10-(Ax,sdx+Am,sdm) for correlating the sample fluorescence measured with a conventional spectrofluorometer and its UV-vis absorbance quantified with a conventional UV-vis spectrophotometer. This model can be simplified or expanded for a variety of fluorescence analyses. First, it enables curve-fitting fluorescence intensity as a function of the fluorophore or sample absorbance over a sample concentration range impossible with existing models. Second, it provides the theoretical foundation for an inner-filter-effect (IFE)-correction method developed earlier and explains mathematically the linearity between the IFE-corrected fluorescence and the fluorophore concentration or absorbance. Third, this model can be expanded for quantitative mechanistic studies of fluorescence intensity variations triggered by stimuli treatments. One demonstrated example is to quantify temperature effects on the emission-wavelength-specific and total fluorescence quantum yield of anthracene. We expect that this first-principle model will be broadly adopted for both student education that promotes evidence-based learning and a variety of fluorescence applications where disentangling sample absorption and emission are critical for reliable data analysis.

Colloidal Polydopamine Beads: A Photothermally Active Support for Noble Metal Nanocatalysts.

Polydopamine (PDA) is a unique bioinspired synthetic polymer that integrates broadband light absorption, efficient photothermal transduction, and versatile surface-adhesion functions in a single material entity. Here, we utilize colloidal PDA beads in the submicron particle size regime as an easily processable and photothermally active support for sub-10 nm Pd nanocatalysts to construct a multifunctional material system that allows us to kinetically boost thermal catalytic reactions through visible and near-infrared light illuminations. Choosing the Pd-catalyzed nitrophenol reduction by ammonium formate as a model transfer hydrogenation reaction exhibiting temperature-dependent reaction rates, we demonstrate that interfacial molecule-transforming processes on metal nanocatalyst surfaces can be kinetically modulated by harnessing the thermal energy produced through photothermal transduction in the PDA supports.

Kinetic spectroscopic quantification using two-step chromogenic and fluorogenic reactions: From theoretical modeling to experimental quantification of biomarkers in practical samples.

Kinetic chromogenic (CG) and fluorogenic (FG) quantification deduces analyte concentration based on the reaction rate between the CG/FG probe and its targeted molecule. Little progress has been made in the past half century in either the theory or the applications of the kinetic spectroscopic quantification methods. Current kinetic CG/FG quantification is limited only to a subset of CG/FG reactions that can be approximated as the single-step process, and more problematically, to research samples with no matrix interferences. Reported herein is a kinetic quantification model established for multistep CG/FG reactions and a proof-of-concept demonstration of direct kinetic FG quantification of biomarkers in practical samples. The kinetic spectral intensity of the CG/FG reactions with two rate-limiting steps comprises three temporal regions: an accelerating period where rate of signal change is increasingly rapid, a linear region where the rate of signal change is approximately constant, and a deceleration region where the rate of signal increase becomes progressively small. Kinetic quantification is performed through simple linear-curve-fitting of the kinetic signal in its linear time-course region. The theoretical model is validated with the dual CG/FG 2-thiobarbituric acid (TBA) and malondialdehyde (MDA) reaction. Proof-of-concept kinetic spectroscopic quantification of analytes in practical samples is demonstrated with the FG quantification of MDA in canned chicken. The only sample preparation is bench-top centrifugation followed by two sequential syringe filtrations. The total kinetic FG assay time is less than 10 min, more than 10 times more efficient than the current equilibrium-based MDA assay. The theoretical model and the measurement design strategies offered by this work should help transform the current kinetic spectroscopic quantification from a niche research tool to an indispensable technique for time-sensitive applications.

Quantification of the Photon Absorption, Scattering, and On-Resonance Emission Properties of CdSe/CdS Core/Shell Quantum Dots: Effect of Shell Geometry and Volumes.

Reliable quantification of the optical properties of fluorescent quantum dots (QDs) is critical for their photochemical, -physical, and -biological applications. Presented herein is the experimental quantification of photon scattering, absorption, and on-resonance-fluorescence (ORF) activities of CdSe/CdS core/shell fluorescent QDs as a function of the shell sizes and geometries. Four spherical QDs (SQDs) with different diameters and four rod-like QDs (RQDs) with different aspect ratios (ARs) have been analyzed using UV-vis, fluorescence, and the recent polarized resonance synchronous spectroscopic (PRS2) methods. All quantum dots are simultaneous absorbers and scatterers in the UV-vis wavelength region, and they all exhibit strong ORF emission in the wavelength regions where the QDs both absorb and emit. The absorption and scattering cross-sections of the CdS shell are linearly and quadratically, respectively, proportional to the shell volume for both the SQDs and RQDs. However, the effects of CdS shell coating on the core optical properties are different between SQDs and RQDs. For RQDs, increasing the CdS shell volume through the length elongation has no effect on either the peak wavelength or intensity of the CdSe core UV-vis absorption and ORF, but it reduces the QD fluorescence depolarization. In contrast, increasing CdS shell volume in the SQDs induces red-shift in the CdSe core peak UV-vis absorption and ORF wavelengths, and increases their peak cross-sections, but it has no effect on the SQD fluorescence depolarization. The RQD ORF cross-sections and quantum yields are significantly higher than their respective counterparts for the SQDs with similar particle sizes (volumes). While these new insights should be significant for the QD design, characterization, and applications, the methodology presented in this work is directly applicable for quantifying the optical activities of optically complex materials where the common UV-vis spectrometry and fluorescence spectroscopy are inadequate.

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

Anisotropy and depolarization are two interconvertible parameters in fluorescence and light scattering spectroscopy that describe the polarization distribution of emitted and scattered photons generated with linearly polarized excitation light. Whereas anisotropy is more frequently used in fluorescence literature for studying association/dissociation of fluorophore-bearing reagents, depolarization is more popular in the light-scattering literature for investigating the effect of scatterers' geometries and chemical compositions. Presented herein is a combined computational and experimental study of the scattering and fluorescence depolarization enhancement induced by light scattering in turbid samples. The most important finding is that sample light scattering and fluorescence depolarization increases linearly with sample light-scattering extinction. Therefore, one can extrapolate the analyte-specific scattering and fluorescence depolarization through linear curve fitting of the sample light scattering and fluorescence depolarization as a function of the sample concentration or the path length of the sampling cuvettes. An example application of this linear extrapolation method is demonstrated for quantifying the fluorophore-specific fluorescence depolarization and consequently its anisotropy for an aggregation-induced-emission sample. This work should be important for a wide range of macromolecular, supramolecular, and nanoscale fluorescent materials that are often strong light scatterers due to their large sizes.

A Divide-and-Conquer Strategy for Quantification of Light Absorption, Scattering, and Emission Properties of Fluorescent Nanomaterials in Solutions.

Optical properties of fluorescent materials including their UV-vis absorption, scattering, and on-resonance fluorescence activities are strongly wavelength-dependent. Reported herein is a divide-and-conquer strategy for experimental quantification of fundamental optical constants of fluorescent nanomaterials including their UV-vis absorption, scattering, and on-resonance-fluorescence (ORF) cross-section spectra and ORF fluorescence and light scattering depolarization spectra. The fluorophore UV-vis extinction spectrum is first divided into a blue and a red wavelength region. The UV-vis extinction cross-section spectrum in the blue wavelength region is decomposed into its absorption and scattering extinction spectra straightforwardly using the established polarized resonance synchronous spectroscopic technique. In its red wavelength region, however, the fluorophores can be simultaneous photon absorbers, scatterers, and anti-Stokes-shifted, on-resonance, and Stokes-shifted fluorescence emitters under the resonance excitation and detection conditions. A polarized anti-Stokes'-shifted, on-resonance, and Stokes'-shifted spectroscopic method is developed for quantifying fluorophore absorption, scattering, one-resonance fluorescence (ORF) cross-section spectra, and scattering and ORF fluorescence depolarization spectra in this wavelength region. Example applications of the presented techniques were demonstrated with fluorescent polystyrene nanoparticles, fluorescent quantum dots, and molecular fluorophores Rhodamine 6G and Eosin Y.

Efficient and scalable high-quality graphene nanodot fabrication through confined lattice plane electrochemical exfoliation.

Reported is a confined lattice plane electrochemical exfoliation method that exploits the electrochemical reaction of face (basal) or side (edge) planes of highly oriented pyrolytic graphite (HOPG) while other planes are blocked using wax, based on the anisotropy of HOPG for efficient and effective fabrication of graphene nanodots with uniform size distribution.

Surface Plasmon Resonance, Formation Mechanism, and Surface Enhanced Raman Spectroscopy of Ag+-Stained Gold Nanoparticles.

A series of recent works have demonstrated the spontaneous Ag+ adsorption onto gold surfaces. However, a mechanistic understanding of the Ag+ interactions with gold has been controversial. Reported herein is a systematic study of the Ag+ binding to AuNPs using several in-situ and ex-situ measurement techniques. The time-resolved UV-vis measurements of the AuNP surface plasmonic resonance revealed that the silver adsorption proceeds through two parallel pseudo-first order processes with a time constant of 16(±2) and 1,000(±35) s, respectively. About 95% of the Ag+ adsorption proceeds through the fast adsorption process. The in-situ zeta potential data indicated that this fast Ag+ adsorption is driven primarily by the long-range electrostatic forces that lead to AuNP charge neutralization, while the time-dependent pH data shows that the slow Ag+ binding process involves proton-releasing reactions that must be driven by near-range interactions. These experimental data, together with the ex-situ XPS measurement indicates that adsorbed silver remains cationic, but not as a charged-neutral silver atom proposed by the anti-galvanic reaction mechanism. The surface-enhanced Raman activities of the Ag+-stained AuNPs are slightly higher than that for AuNPs, but significantly lower than that for the silver nanoparticles (AgNPs). The SERS feature of the ligands on the Ag+-stained AuNPs can differ from that on both AuNPs and AgNPs. Besides the new insights to formation mechanism, properties, and applications of the Ag+-stained AuNPs, the experimental methodology presented in this work can also be important for studying nanoparticle interfacial interactions.

Optical Properties and Kinetics: New Insights to the Porphyrin Assembly and Disassembly by Polarized Resonance Synchronous Spectroscopy.

With their unique photochemical properties, porphyrins have remained for decades the most interested chemicals as photonic materials for applications ranging from chemistry, biology, medicine, to photovoltaic. Porphyrins can self-assemble into higher order structures. However, information has been scant on the kinetics and structural evolution during porphyrin assembly and disassembly. Furthermore, quantitative understanding of the porphyrin optical activities is complicated by the complex interplay of photon absorption, scattering, and fluorescence emission that can concurrently occur in porphyrin samples. Using meso-tetrakis(4-sulfonatophenyl)porphyrin as the model molecule, reported herein is a combined UV-vis extinction, polarized Stokes-shifted fluorescence, and polarized resonance synchronous spectroscopic (PRS2) study of porphyrin assembly and disassembly in acidic solutions. Although porphyrin assembly and disassembly occur instantaneously upon the sample preparation, both processes last at least a few months before reaching their approximate equilibrium states. The two processes were monitored in situ by quantifying the porphyrin fluorescence and scattering depolarizations as well as its extinction, absorption, scattering, and fluorescence emission cross sections. In addition to a series of new insights to the porphyrin assembly and disassembly, the methodology described in this work opens the door for the in situ study of the structural and optical properties of photonic materials comprising molecular assembly.

Synthesis of C-Unsubstituted 1,2-Diazetidines and Their Ring-Opening Reactions via Selective N-N Bond Cleavage.

C-Unsubstituted 1,2-diazetidines, a rarely studied type of four-membered heterocyclic compounds, were synthesized through an operationally simple intermolecular vicinal disubstitution reaction. 1,2-Diazetidine derivatives bearing various N-arylsulfonyl groups were readily accessed and studied by experimental and computed Raman spectra. The ring-opening reaction of the diazetidine was explored and resulted in the identification of a selective N-N bond cleavage with thiols as nucleophiles, which stereoselectively produced a new class of N-sulfenylimine derivatives with C-aminomethyl groups.

Scattering and absorption differ drastically in their inner filter effects on fluorescence, resonance synchronous, and polarized resonance synchronous spectroscopic measurements.

The sample inner filter effect (IFE) induces spectral distortion and affects the linearity between intensity and analyte concentration in fluorescence, Raman, surface enhanced Raman, and Rayleigh light scattering measurements. Existing spectrofluorometric-based measurements treat light scattering and absorption identically in their sample IFEs. Reported herein is the finding that photon scattering and absorption differ drastically in inducing the sample IFE in Stokes-shifted fluorescence (SSF) spectrum, resonance synchronous spectrum (RS2), and the polarized resonance synchronous spectrum (PRS2) measurements. Absorption with an absorption extinction as small as 0.05 imposes significant IFE on SSF, RS2, and PRS2 measurements. However, no significant IFE occurs even when the scattering extinction is as high as 0.9. For samples that both absorb and scatter light, one should decompose their UV-vis extinction spectra into absorption and scattering extinction component spectra before correcting the sample IFE. An iteration PRS2 method was introduced for the experimental decoupling of the photon absorption and scattering contribution. The methodology presented in this work can be easily implemented by researchers with access to one conventional UV-vis spectrophotometer and one spectrofluorometer equipped with a pair of excitation and detection polarizers. This work should be of broad significance in chemical research given the popularity of fluorescence spectroscopy in material characterization applications.