Applied Spectroscopy

To understand the uptake into the body of bioactive “healthy” molecules from fruits and vegetables it is necessary to know where they are located within the original plant structures and what path they take as the plant is processed before consumption. This means not only the location but also the physical state of the molecule, e.g., crystalline or solvated, as this affects uptake. This study demonstrates how confocal Raman microspectroscopy can be used to follow changes in the physical state of carotenoids in tomatoes. We show how the three main carotenoids found in tomatoes, lycopene, β-carotene, and lutein, are distinguished from each other and how their physical state can be determined. We then show how this information can be used in situ within tomato fruit organelles, in three different tomato varieties, to locate the different carotenoids and determine their physical status. The effect of processing on carotenoids in tomatoes is also shown. This type of information can be of great benefit in availability trials to help rationalize results and define the state in which foods should be presented to the body to maximize uptake.

Structural disorder induced by cryogenic milling and by heating to the amorphous phase in the active pharmaceutical ingredient Griseofulvin has been studied using Raman spectroscopy, X-ray powder diffraction (XRPD), and fluorescence spectroscopy. A broad, exciting-frequency-independent scattering background in the Raman spectra and changes in intensities and splitting of some of the Raman lines due to lattice and molecular modes have been observed. In the cryomilled samples this strong background is deconvoluted into two components: one due to lattice disorder induced by cryomilling and the other due to Mie scattering from nanosized crystallites. A single-component background scattering attributed to lattice disorder is seen in the Raman spectrum of the amorphous sample. Fluorescence measurements showed an intrinsic fluorescence signal in as-received Griseofulvin that does not correspond to the inelastic background in the Raman spectra and, moreover, decreases in intensity upon cryomilling, thus excluding an assignment of the Raman background intensity to impurity- or molecular-defect-induced fluorescence. Wide-angle XRPD measurements on cryomilled Griseofulvin shows a broad two-component background consistent with the background-scattering component in the Raman data associated with lattice disorder, but at longer correlation lengths. Persistence of this disorder to even longer lengths is evident in small-angle synchrotron XRPD data on micronized Griseofulvin taken as a function of temperature from the crystalline to the amorphous phase.

Surface-enhanced Raman spectroscopy (SERS) has been shown to be an effective technique for increasing the detection sensitivity in chemical and biological applications. SERS has a distinct advantage over normal Raman spectroscopy, with enhancements typically greater than 10⁴ over the normal Raman signal; however, this advantage in sensitivity comes with a caveat: controlling the spectroscopic reproducibility and enhancement activity of metal nanostructured substrates can be difficult. We present a survey and subsequent data analysis performed on several nanostructured substrates designed for SERS, including silver and gold colloids, silver nanorods, gold nanoshells, and commercially manufactured gold nanostructures.

Metal–organic semiconductor films are fabricated as co-evaporated films. It is demonstrated that the technique can be used to fabricate metal-semiconductor surface-enhanced Raman scattering (SERS) or surface-enhanced resonance Raman scattering (SERRS) substrates or to attain SERS or SERRS of highly insoluble pigments. The experiments are carried out using three different pigments: iron phthalocyanine (FePc) and two substituted tetracaboxylic perylenes (PTCDs). The structure of the co-PVD films was characterized at the nanoscale by scanning electron microscopy through field-emission gun (SEM-FEG), revealing a fairly homogeneous spatial distribution of the silver-pigment nanoparticles. The fabricated mixed nanostructures show a homogenous distribution of plasmon enhancement as observed in the point-by-point mapping of the SERRS spectra recorded with micrometer spatial resolution in Raman microscopy.

The use of surface-enhanced Raman scattering (SERS) for detecting domoic acid and saxitoxin was demonstrated and vibrational modes have been assigned based on the current literature. Silver nanoparticles were used to obtain the SERS spectra of domoic acid for the first time, which displayed enhancement of nearly 70 times the normal Raman spectra. Unique features in the SERS spectrum of domoic acid allowed the binding effect and orientation of the domoic acid to the metal surface to be analyzed. Saxitoxin exhibited an undetectable normal Raman signal but revealed very prominent SERS peaks. SERS peak positions closely matched published experimental and theoretical values, but with different peak ratios, indicating variance in molecule–nanoparticle interaction depending on the SERS substrate utilized. SERS is demonstrated as a powerful analytical tool for detecting toxins at low concentration with molecular specificity and shows immense potential for fast and remote sensing of toxins in various applications.

Resonance Raman microspectroscopy in combination with hierarchical cluster analysis (HCA) is one of the most promising tools for the rapid examination of complex biological and medical samples. HCA is a ready, computerized tool for examining large sets of data for common characteristics, and a multitude of algorithms for this purpose have been developed over the years. However, resonance Raman spectra obtained from complex biological samples may originate from different chromophores as well as from a common chromophore found in different host environments, i.e., bacteria. Therefore, algorithms applied to resonance Raman spectra must handle data of high intrinsic similarity, i.e., spectra originating from a common chromophore, and data with highly dissimilar features, i.e., spectra from different chromophores, in the same unsupervised analysis. We examined the performance of eight widely used algorithms for hierarchical cluster analysis in clustering resonance Raman spectra of bacteria: Single-Linkage (Nearest-Neighbor), Complete-Linkage (Farthest-Neighbor), Average-Linkage, Weighted-Average-Linkage, Centroid, Median, and the Ward algorithm. Algorithm performance was evaluated by comparing the results of clustering a set of high-quality reference spectra with the results obtained when clustering a set of spectra recorded from single cells. References were formed by averaging 100 spectra of individual cells. While all algorithms returned highly similar results when clustering the reference spectra, their performance differed significantly when applied to single spectra. The best-performing algorithm, Weighted-Average-Linkage, correctly grouped single spectra with a reliability of above 95% while the spectral distances between the clusters deviated less than 10% from the results obtained with reference spectra. In contrast, the algorithm performing worst showed no similarity to the reference clustering at all. The widely used Ward algorithm deviated up to 30% from the reference in the spectral distances and returned a different spectral relation between bacteria expressing the same chromophore.

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractal-like silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

Process Raman, infrared (IR), and nuclear magnetic resonance (NMR) analyses are currently being performed in industrial settings for the monitoring of large scale reactions. These methods offer a distinct set of advantages such as no sample preparation and rapid noninvasive remote analysis. Process Raman spectroscopy offers information pertaining to the molecular backbone as well as symmetrical non-polar groups. IR spectroscopy yields information pertaining to hydrogen bonding and asymmetric polar groups. NMR spectrometry provides highly resolved information detailing specific proton environments. These distinct spectral characteristics present a unique opportunity to join together the Raman, IR, and NMR spectra to give one set of “fused” spectra containing complementary information from two sources (Raman and IR) and one orthogonal source (NMR) that describe an industrial process. Data fusion enables process modeling and control to be performed using a single data set. This study has applied the concept of data fusion to characterize a series of crude oil fractions. After collection, the respective spectra were scaled and fused together to form one contiguous spectrum. The multivariate models built using the fused data had a root mean square error of prediction (RMSEP) of 0.307%, a significant reduction in the prediction errors when compared to models built using the separate spectra. The use of data fusion with multiple analytical measurements reduces the error associated with inferential property models for industrial process monitoring, thus allowing for increased understanding and control of an industrial process.

A handheld near-infrared (NIR) analyzer was used to build partial least squares (PLS) models relating jet fuel properties to the spectral data (880–1570 nm). The properties included: API gravity, % aromatics, cetane index, density, 10%/50%/90% recovered distillation temperatures, freezing point, flashpoint, hydrogen content, and % saturates. The models were validated using leave-one-out validation of the training sets and by predicting an independent test set of fuels. The repeatability of the developed NIR methods was determined by performing replicate measurements on a single master analyzer. The reproducibility of the NIR methods was determined by performing measurements on multiple samples using five different analyzers. The results indicate that the NIR methods are useful for rapid monitoring of jet fuel quality at commercial airports during transport, storage, and distribution.

Many pharmaceutical problems require chemical identification of the ingredients present in a drug product, e.g., a tablet. Examples include the identification of the compounds present in many steps of the manufacturing process and the chemical characterization of counterfeit and third-party tablets. Hyperspectral unmixing of near-infrared images is a key method for solving the above problems, as it provides estimates of the number of pure compounds present in a mixture, their spectral signatures, and the corresponding spatially mapped abundance fractions. The performance of hyperspectral unmixing depends upon the degree of homogeneity of the tablets, as well as the pixel resolution used for image acquisition. This work explores the use of the recent simplex identification via split augmented Lagrangian (SISAL) algorithm to unmix near-infrared images of tablets under different homogeneity and pixel resolution conditions. SISAL is known to solve complex problems beyond the reach of previous hyperspectral unmixing methods. The tablets used in this study are 4- and 5-compound model pharmaceutical mixtures, produced with good and poor blending processes, and the acquisition was performed at three pixel resolutions: 8.1, 27.9, and 40.3 μm/pixel. Heterogeneity proved to increase SISAL's accuracy, as did increased pixel resolution in homogeneous tablets. Given the fast image acquisition and algorithm execution times, low- and high-resolution images should always be acquired; combined with the homogeneity grade of the samples, this may be determinant to a case-by-case decision on the proper action to be taken next.

A prototype small photothermal deflection apparatus was constructed on a laboratory bench. Not including excitation laser, data collection computer, and gas pumps, the apparatus fits on a bench space with a footprint of about 10 cm × 10 cm. The apparatus lends itself to miniaturization in future assemblies. The apparatus was tested relative to the conventional laboratory-scale photothermal apparatus. Digital filtering procedures developed in this laboratory were used to collect and analyze the data. Numerical simulation of photothermal signal is performed to accurately predict the heat flow process as sample volumes are scaled down. The results show that increased sensitivity is possible with small apparatus configurations. The prototype apparatus also exhibits high linear response with pulsed irradiance for linear absorbers. Future improvements could include miniaturization with robust configuration that will allow for portable use in trace analysis monitoring of atmospheric pollutants.

Steady-state diffuse reflection spectroscopy is a well-studied optical technique that can provide a noninvasive and quantitative method for characterizing the absorption and scattering properties of biological tissues. Here, we compare three fiber-based diffuse reflection spectroscopy systems that were assembled to create a light-weight, portable, and robust optical spectrometer that could be easily translated for repeated and reliable use in mobile settings. The three systems were built using a broadband light source and a compact, commercially available spectrograph. We tested two different light sources and two spectrographs (manufactured by two different vendors). The assembled systems were characterized by their signal-to-noise ratios, the source-intensity drifts, and detector linearity. We quantified the performance of these instruments in extracting optical properties from diffuse reflectance spectra in tissue-mimicking liquid phantoms with well-controlled optical absorption and scattering coefficients. We show that all assembled systems were able to extract the optical absorption and scattering properties with errors less than 10%, while providing greater than ten-fold decrease in footprint and cost (relative to a previously well-characterized and widely used commercial system). Finally, we demonstrate the use of these small systems to measure optical biomarkers in vivo in a small-animal model cancer therapy study. We show that optical measurements from the simple portable system provide estimates of tumor oxygen saturation similar to those detected using the commercial system in murine tumor models of head and neck cancer.

A quantitative method, based solely on optical absorption, to determine the total iron (Fe) concentration in Fe : LiNbO₃ is proposed. Absorption spectra of several samples doped by thermal diffusion with different concentrations and different [Fe²⁺]/[Fe³⁺] ratios show an isosbestic point at 342 nm. At this wavelength the absorption is proportional to the total Fe concentration and does not depend on the oxidation state. Thanks to the large number of samples covering a wide range of concentrations, in this work it was possible to estimate an effective absorption cross-section relating the absorbance of a given sample to its iron content. The main advantage of the proposed method is in its simplicity and the fact that the result does not depend on the reduction degree of the sample. As it is known that the absorbance of Fe : LN at another wavelength (532 nm) gives information on the amount of Fe²⁺ present in the sample, our method makes it possible to characterize both the total Fe amount and its reduction degree within a single optical absorption measurement.

Far-ultraviolet (FUV) spectra of n-alkanes (n = 5–14) and branched alkanes were measured in their liquid state by using a newly developed attenuated total reflection (ATR)-FUV spectrometer to investigate spectra–structure relationship in the FUV region. The n-alkanes show a broad band near 8.3 eV and a weak shoulder near 7.7 eV. The 8.3 eV band shows a lower energy shift with a significant intensity increase with the increase in the length of alkyl chain. We have assigned the 8.3 eV band to the overlap of two bands due to the transition from the highest occupied molecular orbital (HOMO) to 3p and that from the HOMO-1 to 3s based on the observation that the peak energy of the 8.3 eV band of the n-alkanes is proportional to the first ionization energy. The 7.7 eV shoulder may be due to the transition from HOMO to 3s. The intensity of the 7.7 eV band increases markedly in the order of alkanes without branch, with tertiary, and with quaternary carbon atoms. It is very likely that the forbidden transition from HOMO to 3s becomes allowed by the large decrease in symmetry upon going from the n-alkanes to the branched ones with the quaternary carbon, respectively.

Ultraviolet (UV) spectroscopy has been widely used in monitoring water and wastewater treatment. In this study UV spectroscopy was used to investigate fouling development on the membrane surface of membrane bioreactors. The chemistry of mixed liquor present in the membrane bioreactor and the foulant deposited on the membrane surface was compared by analyzing the UV spectra. The mixed liquor showed different spectra than did the foulant. The foulant spectra showed a shift in absorbance peaks with operation time. The particle size distribution (<450 nm) was also examined to explain the UV fingerprints.

A method is proposed for automatically determining the upper and lower boundaries of the Raman scatter peak in fluorescence spectroscopy from empirical data. Accurate peak boundaries are needed to calculate accurate Raman peak areas, used for normalizing fluorescence signals to produce data in units that are comparable between instruments. Comparisons of Raman peak boundaries across nine individual instruments (FluoroMax 3 (FM3) fluorometers from HORIBA Jobin Yvon and Cary Eclipse (CE) fluorometers from Varian Inc.) at the excitation wavelength λ_ex = 350 nm reveal consistent results. At 350 nm excitation, the Raman peak was confined by the emission wavelengths of 382–418 nm, with boundaries determined for the FM3 fluorometers deviating by no more than 0.5 nm and 1.5 nm with respect to the start and end of the peak, and CE fluorometers deviating by up to 1.5 nm and 2 nm, respectively. Peak width was a function of fluorometer type and excitation wavelength. For the FM3 instruments, widths increased from approximately 30 nm at λ_ex = 300 nm to 40 nm at λ_ex = 380 nm, while for the CE instruments, peaks were approximately 5–8 nm narrower. Code for implementing the procedure in MATLAB, which allows for the adjustment of input parameters to compensate for noisy data, is provided in the Supplemental Material (available online).

Rapid quantitative analyses of powder samples available in tiny amounts have successfully been conducted by utilizing a transversely excited atmospheric (TEA) CO₂ laser-induced He gas plasma. In this study, 4 mg of powder sample was homogeneously mixed with 4 mg of high-vacuum silicon grease and the silicon grease–mixed powder sample (SMP) was painted on a metal surface, which serves as a subtarget. The grease functions to strongly bind the powder and to suppress blow-off of the powder particles. When a TEA CO₂ laser (750 mJ, 10.6 μm, 200 ns) was directly focused on the metal subtarget in He gas at 1 atmosphere, a hightemperature He gas plasma was induced, producing a profusion metastable He atoms. It is assumed that the powder particles together with the silicon grease were vaporized to be effectively atomized and excited through metastable He atoms. The result revealed that this technique can be widely employed in the rapid semi-quantitative analyses of powder samples present in minute amounts. A quantitative analysis of loam soil containing different concentrations of Cu was successfully demonstrated, resulting in a good linear calibration curve. The detection limits of Cr and Pb in loam soil were approximately 4 and 13 mg/kg, respectively. Also, we confirmed that this technique can be applied to check the quality of commercial products such as gold film (Au foil), mineral supplement tablets, and prestigious cosmetic powders.