Correction of inner-filter effect in fluorescence excitation-emission matrix spectrometry using Raman scatter

Fluorescence excitation-emission matrix (EEM) spectroscopy is a useful tool for interpretation of fluorescence information from natural water samples. One of the major problems with this technique is the inner-filter effect (IFE), i.e. absorption of light at both the excitation and emission wavelengths. The common solutions are to either dilute the sample or apply some form of mathematical correction, most often based on the measured absorbance of the sample. Since dilution is not always possible, e.g. in on-line or in situ EEM recordings, and corrections based on absorbance are hampered primarily by the use of a separate absorbance instrument, neither of these solutions is optimal. In this work, we propose a mathematical correction procedure based on the intensity of Raman scatter from water. This procedure was found to reduce the error after correction by up to 50% in comparison with two absorbance correction procedures. Furthermore, it does not require the use of a separate absorbance measurement, and it is applicable to on-line and in situ EEM recordings, where the IFE would otherwise cause problems.     

Multivariate curve resolution analysis excitation-emission matrices of fluorescence of humic substances

Excitation-emission matrices (EEM) of fluorescence of aqueous solutions of humic substances (HS), and sets of EEM acquired as function of the HS concentration, were analysed by multivariate curve resolution alternating least squares (MCR-ALS). Three types of HS samples were studied: one commercial humic acid; two samples of fulvic acid (FA) extracted from a pinewood soil; two samples of FA extracted from recycled wastes. The fluorescence measurements were carried out at HS concentration between 5 and 100 mg/L and at pH 6. The application of MCR-ALS algorithm on each individual EEM, as well as on column-wise augmented matrices, allows the identification of three major fluorophores in all HS samples analysed. The emission and excitation spectra of these fluorophores were recovered and are characteristic of each sample. Moreover, the variation of the fluorescence intensities of each fluorophore with HS concentration shows deviations from linearity at HS concentration higher than 30 mg/L, depending on the fluorophore and/or sample. This behaviour reveals the existence of inner filter effects that affect the proportionally between the fluorescent signal and concentration but do not provoke measurable distortions on the fluorescence spectra of the detected fluorophores.     

Spectroscopic diagnosis of colonic dysplasia.

We have developed a method for defining diagnostic algorithms for pathologic conditions based on fluorescence spectroscopy. We apply this method to human colon tissue and show that fluorescence can be used to diagnose the presence or absence of colonic adenoma. This method uses fluorescence excitation-emission matrices (EEM) to identify optimal excitation regions for obtaining fluorescence emission spectra which can be used to differentiate normal and pathologic tissues. In the case of normal and adenomatous colon tissue, these were found to be: 330, 370, and 430 nm +/- 10 nm. At these excitation wavelengths, emission wavelengths for use in diagnostic algorithms are identified from average difference and ratio of the spectra from normal and pathologic tissues. In colon tissue, at 370 nm excitation, 404, 480, and 680 nm were found to be useful emission wavelengths for diagnosing the presence of adenoma in vitro. The basis of colon tissue autofluorescence was investigated using EEM of pure molecules and relevant excitation-emission maxima in the literature.     

A light scattering and fluorescence emission coupled ratiometry using the interaction of functional CdS quantum dots with aminoglycoside antibiotics as a model system

Any signals, if their intensities have simple functional relationship with analyte concentration, can be applied to analytical purposes. Rayleigh light scattering signals and fluorescence signals are twins in flurospectroscopy, so the light scattering signals are the major interference when the Stokes shift is small. Herein, we propose a light scattering and fluorescence emission (LS-FL) coupled ratiometry using CdS quantum dots (QDs) as a fluorescence probe to detect aminoglycoside antibiotics (AGs). As model analytes, AGs, when attached to the surface of CdS-QDs via electrostatic interaction in aqueous medium, result in strong enhanced light scattering (LS) emission characterized at 376 nm and fluorescence quenching of CdS-QDs at 500 nm. Thus, a ratiometry using the coexistent light scattering and fluorescent emission signals has been proposed. Based on the linear relationship between logarithm of light scattering and fluorescence emission ratio (R) and logarithm of AGs concentration, a novel assay of AGs is established with the limits of detection (3σ) being 58-190 nmol l⁻¹, and applied successfully to detect AGs injection and serum samples.     

Contributions to accuracy improvement of simultaneous ICP atomic emission spectrometry using multi-line measurements of analyte and internal standard elements Applications for the analysis of permalloy

For successful application of simultaneous ICP atomic emission spectrometry for major component determinations in multi-component materials the accuracy of the method has to be improved. As a contribution to solve this problem a combined procedure for multi-component standard sample preparation, optimum calibration and different variations of internal standard corrections is described. Variance-weighted multi-line calibrations give most accurate results. Internal standard corrections are effective, if the time-dependent spectral line intensity fluctuations of the standard and the analyte elements are well correlated. Their sensitivities against some responsible device parameter variations are investigated. On the basis of multi-line measurements of the analyte and internal standard elements a “group-selected internal standard correction” (GS-ISC) method is applied and results in relative errors of less than 1% even for extreme fluctuations of the raw intensities. For rapid routine determination methods of materials with variable element compositions the added line intensities of the internal standard element can be used to correct the added analyte line raw intensities (“intensity addition internal standard correction” (IA-ISC) method). These accuracy optimization procedures are applied for the analysis of the soft magnetic material permalloy using the internal standard element In.     

Correction for non-spectroscopic matrix effects in inductively coupled plasma-atomic emission spectroscopy by internal standardization using spectral lines of the same analyte

The common analyte internal standardization (CAIS) technique was extended to correct for non-spectroscopic matrix effects in inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurements. The technique is based on simultaneous measurement of two different spectral lines of the same analyte. A matrix correction factor is then estimated from its linear correlation with the ratio of intensities of these two measured lines. Experimental tests with four elements (Ba, La, Mg, and Mn) in three matrices (NaCl, H₂SO₄, HNO₃) demonstrate a significant decrease (from 3 to 22 times) of the matrix effect after correction.     

Fluorimetric determination of amphetamine in urine by flow injection with on-line liquid–liquid extraction

Determination of amphetamine in urine was performed by batch and flow injection methodologies. The suitable experimental conditions for fluorimetric measurements were established. The liquid–liquid extraction was carried out at pH 13 using diethyl ether as extracting reagent. The measurement conditions were 260 nm and 277 nm for excitation and emission wavelengths, respectively. The method requires standard addition calibration and Youden blank correction. The influence of the main metabolites of amphetamine and metamphetamine were studied. The accuracy and precision of the proposed method was tested and the method is adapted to the flow injection procedure with on-line extraction.     

Assessment of the oxidative stability of lubricant oil using fiber-coupled fluorescence excitation–emission matrix spectroscopy

The fluorescence of antioxidant additives in lubricant oil was used as an indicator of oxidative stability of the oil. It was found that the decrease in fluorescence intensities of phenyl-α-napthylamine, its dimer, and another unidentified antioxidant coincide with the formation of decomposition products of the oil base stock. Simple kinetic models were developed that were capable of describing antioxidant reactions as a pseudo first-order processes. It is shown that fluorescence excitation emission matrix (EEM) spectroscopy coupled with an optical fiber probe can provide real-time assessment of the oxidative stability of the lubricant. Parallel factor (PARAFAC) analysis was used to correlate the component scores to the oil breakdown number.     

A study of non-linear calibration graphs for brass with femtosecond laser-induced breakdown spectroscopy

The ablation of brass samples with 170 fs laser pulses was studied by time resolved emission spectroscopy. The intensities of Cu as well as of Zn lines were found to be non-linearly dependent on their concentrations in the samples. This behaviour is explained by changes in the mass ablation rate. Thus, linear dependencies were obtained when the Zn line intensities were normalized to the line intensities of Cu. A simple theoretical model based on compositional differences is presented. The differences in ablation are accounted for using the standard two-temperature diffusion model for short laser pulse ablation and the steady current resistivity as a fundamental parameter describing the compositional changes in a set of brass samples. In principle, it is possible to construct linear calibration curves from the measured Zn and Cu line intensities without internal standardization if the model and energy dependent ablation measurements are taken into account. Inductively coupled plasma mass spectrometry measurements are used to demonstrate this.     

Calibration transfer employing univariate correction and robust regression

This paper proposes a new method for calibration transfer, which was specifically designed to work with isolated variables, rather than the full spectrum or spectral windows. For this purpose, a univariate procedure is initially employed to correct the spectral measurements of the secondary instrument, given a set of transfer samples. A robust regression technique is then used to obtain a model with low sensitivity with respect to the univariate correction residuals. The proposed method is employed in two case studies involving near infrared spectrometric determination of specific mass, research octane number and naphthenes in gasoline, and moisture and oil in corn. In both cases, better calibration transfer results were obtained in comparison with piecewise direct standardization (PDS). The proposed method should be of a particular value for use with application-targeted instruments that monitor only a small set of spectral variables.     

A fully robust PARAFAC method for analyzing fluorescence data

Parallel factor analysis (PARAFAC) is a widespread method for modeling fluorescence data by means of an alternating least squares procedure. Consequently, the PARAFAC estimates are highly influenced by outlying excitation–emission landscapes (EEM) and element‐wise outliers, like for example Raman and Rayleigh scatter. Recently, a robust PARAFAC method that circumvents the harmful effects of outlying samples has been developed. For removing the scatter effects on the final PARAFAC model, different techniques exist. Newly, an automated scatter identification tool has been constructed. However, there still exists no robust method for handling fluorescence data encountering both outlying EEM landscapes and scatter. In this paper, we present an iterative algorithm where the robust PARAFAC method and the scatter identification tool are alternately performed. A fully automated robust PARAFAC method is obtained in that way. The method is assessed by means of simulations and a laboratory‐made data set. Copyright © 2009 John Wiley & Sons, Ltd.     

Utilization of PARAFAC‐Modeled Excitation‐Emission Matrix (EEM) Fluorescence Spectroscopy to Identify Biogeochemical Processing of Dissolved Organic Matter in a Northern Peatland

In this study, we contrast the fluorescent properties of dissolved organic matter (DOM) in fens and bogs in a Northern Minnesota peatland using excitation emission matrix fluorescence spectroscopy with parallel factor analysis (EEM‐PARAFAC). EEM‐PARAFAC identified four humic‐like components and one protein‐like component and the dynamics of each were evaluated based on their distribution with depth as well as across sites differing in hydrology and major biological species. The PARAFAC‐EEM experiments were supported by dissolved organic carbon measurements (DOC), optical spectroscopy (UV‐Vis), and compositional characterization by ultrahigh resolution Fourier transform ion cyclotron resonance mass spectroscopy (FT‐ICR MS). The FT‐ICR MS data indicate that metabolism in peatlands reduces the molecular weights of individual components of DOM, and oxygen‐rich less aromatic molecules are selectively biodegraded. Our data suggest that different hydrologic and biological conditions within the larger peat ecosystem drive molecular changes in DOM, resulting in distinctly different chemical compositions and unique fluorescent fingerprints. PARAFAC modeling of EEM data coupled with ultrahigh resolution FT‐ICR MS has the potential to provide significant molecular‐based information on DOM composition that will support efforts to better understand the composition, sources, and diagenetic status of DOM from different terrestrial and aquatic systems.     

Development of FRET-based dual-excitation ratiometric fluorescent pH probes and their photocaged derivatives

Dual-excitation ratiometric fluorescent probes allow the measurement of fluorescence intensities at two excitation wavelengths, which should provide a built-in correction for environmental effects. However, most of the small-molecule dual-excitation ratiometric probes that have been reported thus far have shown rather limited separation between the excitation wavelengths (20-70 nm) and/or a very small molar absorption coefficient at one of the excitation wavelengths. These shortcomings can lead to cross-excitation and thus to errors in the measurement of fluorescence intensities and ratios. Herein, we report a FRET-based molecular strategy for the construction of small-molecule dual-excitation ratiometric probes in which the donor and acceptor excitation bands exhibit large separations between the excitation wavelengths and comparable excitation intensities, which is highly desirable for determining the fluorescence intensities and signal ratios with high accuracy. Based on this strategy, we created a coumarin-rhodamine FRET platform that was then employed to develop the first class of FRET-based dual-excitation ratiometric pH probes that have two well-resolved excitation bands (excitation separations>160 nm) and comparable excitation intensities. In addition, these pH probes may be considered as in a kind of "secured ratioing mode". As a further application of these pH probes, the dual-excitation ratiometric pH probes were transformed into the first examples of photocaged dual-excitation ratiometric pH probes to improve the spatiotemporal resolution. It is expected that the modular nature of our FRET-based molecular strategy should render it applicable to other small-molecule dual-dye energy-transfer systems based on diverse fluorescent dyes for the development of a wide range of dual-excitation ratiometric probes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Detection of verapamil drug by fluorescence and trilinear decomposition techniques

A three-way analytical methodology experimentally based on fluorescence excitation emission matrix (EEM) and in PARAFAC and TLD chemometric analysis was assessed for the quantification of verapamil drug in a tablet formulation. A standard addition procedure generates experimental information compatible with the chemometric data analysis model allowing the estimation of verapamil with a detection limit of about 0.04 mg/l using methanol as solvent. The structure of the verapamil EEM follows a trilinear model, but background signals (first- and second-order scatter bands) did not—a trilinear three-factor model is necessary to describe experimental datasets. The comparison of a three-factor PARAFAC model with a United States Pharmacopoeia (USP) standard chromatographic method showed similar results.     

三峡库区水体溶解有机质的荧光光谱特性

利用荧光发射和三维荧光光谱(EEM)研究了三峡库区(TGRA)长江干流及嘉陵江、乌江两支流水体溶解有机质(DOM)的荧光特性.结合采样段面溶解有机碳(DOC)、氧化还原电位(ORP)、pH等参数,考察了干流、两支流及其汇合后各水体DOM的荧光指数f450/500和类腐殖酸与类富里酸强度比值rC/D变化,分析了类腐殖酸、类富里酸及类蛋白质在库区支流与干流、上游与下游水体的来源、组成、分布及环境行为.实验表明: 干流水体中DOM以类富里酸、类蛋白质荧光有机质为主;嘉陵江水体DOM以类蛋白质为主,富里酸次之; 在朝天门与长江汇合后,类蛋白峰强减弱而类腐殖酸有一定增加;乌江以类富里酸为主,汇合干流后,富里酸和类蛋白质峰增强;库区上游水体DOM主要受嘉陵江影响;干流DOM受陆源性影响.初步揭示了水体DOM的EEM特性与库区水质参数的相关性,为水体监测与分析提供参考.     

Imaging fingerprinting of excitation emission matrices

The spectral fingerprinting of the excitation emission matrix (EEM) of fluorescent substances is demonstrated using polychromatic light sources and tri-chromatic image detectors. A model of the measured fingerprints explaining their features and classification performance, based on the polychromatic excitation of the indicators is proposed.Substantial amount of spectral information is retained in the fingerprints as corroborated by multivariate analysis and experimental conditions that favor such situation are identified.In average, for five different substances, the model shows a fitting goodness measured by the Pearson's r coefficient and the root mean square deviation of 0.8541 and 0.0247 respectively, while principal component classification patterns satisfactorily compare with the EEM spectroscopy classification and respectively explain 96% and 93% of the information in the fist two principal components.The measurements can be performed using regular computer screens as illumination and web cameras as detectors, which constitute ubiquitous and affordable platforms compatible with distributed evaluations, in contrast to regular instrumentation for EEM measurements.     

Fluorescence,Absorption, Chromatography and Structural Transformation of Chelerythrine and Ethoxychelerythrine in Protic Solvents: A Comparative Study

Chelerythrine (CH) and ethoxychelerythrine (ECH) are chemical reference substances for quality control of Chinese herbal medicines, and ECH is the dihydrogen derivative of CH. In this study, their fluorescence and absorption spectra, as well as their structural changes in different protic solvents were compared. It was observed that their emission fluorescence spectra in methanol were almost the same (both emitted at 400 nm), which may be attributed to the nucleophilic and exchange reactions of CH and ECH with methanol molecules with the common product of 6-methoxy-5,6-dihydrochelerythrine (MCH). When diluted with water, MCH was converted into CH, which mainly existed in the form of positively charged CH⁺ under acidic and near-neutral conditions with the fluorescence emission at 550 nm. With the increase of pH value of the aqueous solution, CH⁺ converted to 6-hydroxy-5,6-dihydrochelerythrine (CHOH) with the fluorescence emission at 410 nm. The fluorescence quantum yields of MCH and CHOH were 0.13 and 0.15, respectively, and both the fluorescence intensities were much stronger than that of CH⁺. It is concluded that CH and ECH can substitute each other in the same protic solvent, which was further verified by high-performance liquid chromatography. This study will help in the investigation of structural changes of benzophenanthridine alkaloids and will provide the possibility for the mutual substitution of standard substances in relevant drug testing.     

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).     

Intermolecular interactions in albumin-sulfoxide-water systems at low temperatures, investigated by means of fluorescence quenching

The effect of low temperatures on the interaction in human serum albumin (HSA)-diethyl sulfoxife (DESO)-dipropyl sulfoxide (DPSO)-water systems is investigated by means of fluorescence spectroscopy (intrinsic protein fluorescence, three-dimensional excitation/emission matrix (3D EEM)). The Stern-Volmer constants of HSA quenching are calculated for these systems. The structural changes occurring in these systems are characterized using 3D EEM profiles of HSA. It is shown that the HSA structural changes depend not only on the direct interaction of protein with sulfoxides but on structural changes in the solvent as well.