Showing papers in "Applied Spectroscopy in 2009"

Fluorescence Intensity Calibration Using the Raman Scatter Peak of Water

Abstract: Fluorescence data of replicate samples obtained from different fluorescence spectrometers or by the same spectrometer but with different instrument settings can have great intensity differences. In order to compare such data an intensity calibration must be applied. Here we explain a simple calibration method for fluorescence intensity using only the integrated area of a water Raman peak. By applying this method to data from three different instruments, we show that it is possible to remove instrument-dependent intensity factors, and we present results on a unified scale of Raman units. The method presented is a rapid and simple approach suitable for routine measurements with no need for hazardous chemicals.

Chemical Imaging of Live Cancer Cells in the Natural Aqueous Environment

Abstract: Chemical imaging with Fourier transform infrared (FT-IR) spectroscopy allows the visualization of the distribution of chemical components in cells without the need for labels or added dyes. However, obtaining such images of living cells is difficult because of the strong absorption of water in the mid-infrared region. We report the use of attenuated total reflection (ATR) FT-IR spectroscopic imaging to study live human cancer cells in an aqueous environment, on a single cell level. Two complementary approaches have been used, providing flexibility with field of view and spatial resolution: (1) micro-ATR FT-IR imaging using a microscope objective with a Ge crystal, and (2) single-reflection diamond ATR-FT-IR imaging. Using both approaches, the ATR-FT-IR spectroscopic signatures allow the differentiation between several cellular organelles, e.g., the nucleus and the endoplasmic reticulum (ER). The overall cell shape can be defined by the distribution of the amide II band in the measured image, while the DNA-rich nucleus and glycogen-rich ER could be imaged using the spectral bands at 1084 cm−1 and 1023 cm−1, respectively. We also demonstrate the potential of ATR-FT-IR spectroscopic imaging for unraveling the details of the dynamics of biological processes, which are not accessible from cell ensemble studies, with high molecular specificity and satisfactory spatial resolution.

Transcutaneous Raman Spectroscopy of Murine Bone In Vivo

Abstract: Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice.

An iterative algorithm for background removal in spectroscopy by wavelet transforms.

Abstract: Wavelet transforms are an extremely powerful tool when it comes to processing signals that have very “low frequency” components or non-periodic events. Our particular interest here is in the ability of wavelet transforms to remove backgrounds of spectroscopic signals. We will discuss the case of surface-enhanced Raman spectroscopy (SERS) for illustration, but the situation it depicts is widespread throughout a myriad of different types of spectroscopies (IR, NMR, etc.). We outline a purpose-built algorithm that we have developed to perform an iterative wavelet transform. In this algorithm, the effect of the signal peaks above the background is reduced after each iteration until the fit converges close to the real background. Experimental examples of two different SERS applications are given: one involving broad backgrounds (that do not vary much among spectra), and another that involves single molecule SERS (SM-SERS) measurements with narrower (and varying) backgrounds. In both cases, we will show that wavelet transforms can be used to fit the background with a great deal of accuracy, thus providing the framework for automatic background removal of large sets of data (typically obtained in time-series or spatial mappings). A MATLAB® based application that utilizes the iterative algorithm developed here is freely available to download from http://www.victoria.ac.nz/raman/publis/codes/cobra.aspx.

Confocal Raman microscopy: performance, pitfalls, and best practice.

Abstract: C onfocal Raman microscopy is an extremely useful technique that permits nondestructive, spatially resolved measurements deep within transparent samples simply by focusing the laser beam at the point of interest. Moving the laser focus allows generation of one-dimensional (1D) depth profiles, and 2D and 3D (volumetric) images. However, in order to correctly interpret the data, it is important to understand exactly where the laser beam is focused and to know the volumetric resolution of the probe beam. These are actually non-trivial questions. The objective of this article is to summarize the critical factors that determine the spatial accuracy, resolution, and sensitivity of confocal Raman microscopy and to highlight the precautions that should be taken to collect high quality, quantitative data. No attempt is made to review the applications of Raman microscopy; these are simply too diverse, spanning topics from art conservation to medical diagnosis. However, the same basic principles must be adhered to, irrespective of the application, if reliable conclusions are to be drawn. Two main topics are considered. The first is the need for properly corrected objectives for depth profiling beneath the surface of transparent samples. If this is not done, the confocal profile will have an incorrect depth scale, degraded depth resolution, and reduced spectral intensity and signal-to-noise ratio (S/N). Even if modeling is used to account for the aberrations and to compute corrected profiles, degraded depth resolution and S/N still occur, which limits the performance. The second key issue is that even with a corrected objective operating with the best attainable resolution, the axial point spread function, which determines the depth resolution, has quite broad wings, so weak signals can be detected from regions quite distant (tens of micrometers) from the point of tightest focus. With thick transparent samples, the integrated signal from these out-of-focus domains can be significant or even dominant, resulting in unusual and counterintuitive observations. This effect is noticeable both for confocal profiling and for lateral scanning over cross-sections; in short, one cannot simply assume that data is acquired with a volumetric resolution of ~1 lm. The effect is especially important when one needs to chemically interpret the spectra rather than just view an image or a profile, since this leads to contamination of spectra with spurious bands. Finally, it is important to note that while some of the effects discussed here seem strange when they are first encountered, most have been known since the early days of confocal micro-spectroscopy. Consequently, few of the results discussed here would necessarily surprise a skilled microscopist. However, it is clear from the literature over the last decade or so that many Raman microscopists (the author included) are gradually re-learning these lessons and, as a result, significant advances have been made in the acquisition and interpretation of confocal Raman data. It therefore seems appropriate and timely to summarize these learning points in a review article.

Gold nanoparticle based surface-enhanced Raman scattering spectroscopy of cancerous and normal nasopharyngeal tissues under near-infrared laser excitation.

Abstract: The capabilities of using gold nanoparticle based near-infrared surface-enhanced Raman scattering (SERS) to obtain biochemical information with high spatial resolution from human nasopharyngeal tissue were presented in this paper The gold nanoparticles used have a mean diameter of 43 nm with a standard deviation of 6 nm The SERS bands of nasopharyngeal tissue were assigned to known molecular vibrations of nucleic acids, amino acids, proteins, and metabolites We also observed the blinking phenomenon at the tissue level when measuring the nasopharyngeal tissue SERS spectra, most frequently in signal intensity but also occasionally in peak positions This phenomenon is excitation light intensity dependent This work demonstrated great potential for using SERS imaging for distinguishing cancerous and normal nasopharyngeal tissues on frozen sections without using any dye labeling or other chemical species as functionalized binding sites

Noninvasive Mail Inspection System with Terahertz Radiation

Abstract: Using the high penetrability of the terahertz waves and the characteristic absorption spectra in this frequency range, we have built a noninvasive mail inspection system targeting drugs and explosives. The system is composed of two stages; in the first stage, the scattering of a continuous terahertz wave is used for selecting mail that contains concealed powder; in the second stage, the absorption spectrum of the suspicious mail is measured and the material is identified using a terahertz spectrum database. We evaluated the performance and the limits of the inspection system.

Detection of uranium using laser-induced breakdown spectroscopy.

Abstract: The goal of this work is a detailed study of uranium detection by laser-induced breakdown spectroscopy (LIBS) for application to activities associated with environmental surveillance and detecting weapons of mass destruction (WMD). The study was used to assist development of LIBS instruments for standoff detection of bulk radiological and nuclear materials and these materials distributed as contaminants on surfaces. Uranium spectra were analyzed under a variety of different conditions at room pressure, reduced pressures, and in an argon atmosphere. All spectra displayed a high apparent background due to the high density of uranium lines. Time decay curves of selected uranium lines were monitored and compared to other elements in an attempt to maximize detection capabilities for each species in the complicated uranium spectrum. A survey of the LIBS uranium spectra was conducted and relative emission line strengths were determined over the range of 260 to 800 nm. These spectra provide a guide for selection of the strongest LIBS analytical lines for uranium detection in different spectral regions. A detection limit for uranium in soil of 0.26% w/w was obtained at close range and 0.5% w/w was achieved at a distance of 30 m. Surface detection limits were substrate dependent and ranged from 13 to 150 microg/cm2. Double-pulse experiments (both collinear and orthogonal arrangements) were shown to enhance the uranium signal in some cases. Based on the results of this work, a short critique is given of the applicability of LIBS for the detection of uranium residues on surfaces for environmental monitoring and WMD surveillance.

Near-infrared spectroscopy for the prediction of disease ratings for Fiji leaf gall in sugarcane clones.

Abstract: This paper demonstrates how inferential measurements or indirect methods using near-infrared (NIR) methodology and chemometrics can be used to predict sugarcane clonal performance. Fiji leaf gall resistance is used in this study as an example. Fiji leaf gall is one of Australia's most serious sugarcane diseases, representing a significant problem in almost half of the total area under production. Traditional rating of sugarcane clones for resistance/susceptibility is difficult and expensive because of the nature of field-based methods and variable infection levels of the trials. Thus, the aim of this work was to investigate the potential of NIR spectroscopy as an alternative means to rate clones from direct measurement of sugarcane leaf spectra and to examine its ability to successfully predict traditional resistance ratings using a calibration model based on a chemometrics method such as partial least squares (PLS). A scanning electron microscopy (SEM) study of the leaf substrate was undertaken to elucidate the nature of the NIR sample site. In addition, an NIR study of freeze-dried sugarcane leaf samples resolved the heavily overlapping O–H bands present in the NIR spectrum due to water/cellulose interaction. A significant decrease in the spectral intensity between 5205 and 5393 cm−1 was observed and a similar decrease was noted in the OH stretching overtone (7114 cm−1) with an accompanying shift to lower wavenumbers. PLS modeling based on traditional ratings as the dependent variable and the corresponding NIR spectra showed satisfactory results with standard error of validation (SEV) and standard error of prediction (SEP) values being 0.98 (R2 = 0.97) and 1.20 (R2 = 0.88), respectively. This methodology has now been recommended for more extensive field trials.

Laser-Induced Breakdown Spectroscopy for In-Cylinder Equivalence Ratio Measurements in Laser-Ignited Natural Gas Engines

Abstract: In this contribution we present the first demonstration of simultaneous use of laser sparks for engine ignition and laser-induced breakdown spectroscopy (LIBS) measurements of in-cylinder equivalence ratios. A 1064 nm neodynium yttrium aluminum garnet (Nd:YAG) laser beam is used with an optical spark plug to ignite a single cylinder natural gas engine. The optical emission from the combustion initiating laser spark is collected through the optical spark plug and cycle-by-cycle spectra are analyzed for Hα(656 nm), O(777 nm), and N(742 nm, 744 nm, and 746 nm) neutral atomic lines. The line area ratios of Hα/O777, Hα/N746, and Hα/Ntot (where Ntot is the sum of areas of the aforementioned N lines) are correlated with equivalence ratios measured by a wide band universal exhaust gas oxygen (UEGO) sensor. Experiments are performed for input laser energy levels of 21 mJ and 26 mJ, compression ratios of 9 and 11, and equivalence ratios between 0.6 and 0.95. The results show a linear correlation (R2 > 0.99) of line intensity ratio with equivalence ratio, thereby suggesting an engine diagnostic method for cylinder resolved equivalence ratio measurements.

Application of the Kubelka—Munk Correction for Self-Absorption of Fluorescence Emission in Carmine Lake Paint Layers

Abstract: The variations of the fluorescence emission of carmine lake travelling through an absorbing and scattering medium, such as a paint layer, were investigated by ultraviolet (UV)-visible absorption, fluorescence spectroscopy, and imaging techniques Samples of the lake were studied in dilute and saturated solutions, on a reference test panel and a real case study Relevant spectral modifications have been observed as a function of the lake concentration mainly consisting of a fluorescence quenching, red shift of emission maxima, and deformation of emission band The application of a correction factor based on the Kubelka-Munk model allowed fluorescence spectra obtained in solution and on painted samples of known composition to be compared and correlated, highlighting that the fluorescence of the lake within paint layers is affected by both self-absorption and aggregation phenomena This approach has been successfully applied on a painting by G Vasari for the noninvasive identification of carmine lake The results reported here emphasize the necessity of taking physical phenomena into account in the interpretation of the fluorescence spectra for a proper and reliable characterization and identification of painting materials in works of art

Emission enhancement of laser-induced breakdown spectroscopy by localized surface plasmon resonance for analyzing plant nutrients.

Abstract: We demonstrate the monitoring of plant nutrients in leaves of Citrus unshiu and Rhododendron obtusum using low-energy (<1 mJ) laser-induced breakdown spectroscopy. The raw plant leaf was successfully ablated without desiccation before laser irradiation, by applying metallic colloidal particles to the leaf surface. The emission intensity with the metallic particles was larger than that without the particles. This result indicates an improvement of the sensitivity and the detection limit of laser-induced breakdown spectroscopy. The emission enhancement was caused by localized surface plasmon resonance and was dependent on the size and material of metallic particles.

Quantitative surface-enhanced Raman spectroscopy based analysis of microRNA mixtures.

Abstract: We have developed a rapid, sensitive, and quantitative method for identification of microRNA (miRNA) sequences in multicomponent mixtures using surface-enhanced Raman spectroscopy (SERS). The method uses Ag nanorod array substrates prepared by oblique angle vapor deposition as the SERS platform. We show that Ag nanorod-based SERS spectra are uniquely characteristic for each miRNA sequence studied, and that the spectral reproducibility is sufficient for quantitative analysis of miRNA profiles in multicomponent mixtures using partial least squares (PLS) regression analysis. This method was applied to individual sample mixtures consisting of two, three, and five miRNAs. Separate PLS models were generated for the two-, three-, and five-component mixtures from >150 calibration spectra covering a concentration range of 6 to 150 μM for each miRNA. The PLS models were externally validated with independent test samples resulting in root mean square errors of prediction (RMSEP) of 7.4, <7.4, and <10 μM for the two-, three-, and five-component models, respectively. These results demonstrate the applicability of SERS for quantitative detection and profiling of miRNAs and suggest that SERS may prove to be a novel, label-free method for identification of disease biomarkers.

Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens.

Abstract: This study deals with the rapid detection and differentiation of Escherichia coli, Salmonella, and Campylobacter, which are the most commonly identified commensal and pathogenic bacteria in foods, using fluorescence spectroscopy and multivariate analysis. Each bacterial sample cultured under controlled conditions was diluted in physiologic saline for analysis. Fluorescence spectra were collected over a range of 200-700 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. The synchronous scan technique was employed to find the optimum excitation (lambda(ex)) and emission (lambda(em)) wavelengths for individual bacteria with the wavelength interval (Deltalambda) being varied from 10 to 200 nm. The synchronous spectra and two-dimensional plots showed two maximum lambda(ex) values at 225 nm and 280 nm and one maximum lambda(em) at 335-345 nm (lambda(em) = lambda(ex) + Deltalambda), which correspond to the lambda(ex) = 225 nm, Deltalambda = 110-120 nm, and lambda(ex) = 280 nm, Deltalambda = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the fluorescence spectra resulted in successful classification of the bacteria by their genus as well as determining their concentration. The detection limit was approximately 10(3)-10(4) cells/mL for each bacterial sample. These results demonstrated that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to classify bacterial pathogens in liquids. The methodology is rapid (>10 min), inexpensive, and requires minimal sample preparation compared to standard analytical methods for bacterial detection.

Calibration Maintenance and Transfer Using Tikhonov Regularization Approaches

Abstract: Maintaining multivariate calibrations is essential and involves keeping models developed on an instrument applicable to predicting new samples over time. Sometimes a primary instrument model is needed to predict samples measured on secondary instruments. This situation is referred to as calibration transfer. This paper reports on using a Tikhonov regularization (TR) based method in both cases. A distinction of the TR design for calibration maintenance and transfer is a defined weighting scheme for a small set of new (transfer or standardization) samples augmented to the full set of calibration samples. Because straight application of basic TR theory is not always possible with calibration maintenance and transfer, this paper develops a generic solution to always enable application of TR. Harmonious (bias/variance tradeoff) and parsimonious (effective rank) considerations for TR are compared with the same TR format applied to partial least squares (PLS), showing that both approaches are viable solutions to the calibration maintenance and transfer problems.

Optical Interference Effects in the Design of Substrates for Surface-Enhanced Raman Spectroscopy

Abstract: This paper presents results showing that the design of substrates used for surface-enhanced Raman spectroscopy (SERS) can impact the apparent enhancement factors (EFs) obtained due to optical interference effects that are distinct from SERS, providing additional enhancement of the Raman intensity. Thus, a combination of SERS and a substrate designed to maximize interference-based enhancement is demonstrated to give additional Raman intensity above that observed for SERS alone. The system explored is 4-nitroazobenzene (NAB) and biphenyl (BP) chemisorbed on a nanostructured silver film obtained by vacuum deposition of Ag on thermally oxidized silicon wafers. The enhancing silver layer is partially transparent, enabling a standing wave to form as a result of the combination of the incident light and light reflected from the underlying Si substrate (i.e., light that passes through the Ag and the intervening dielectric layer of SiO(x)). The Raman intensity is measured as a function of the thickness of the thermal oxide layer in the range from approximately 150 to approximately 400 nm, and despite a lack of morphological variation in the silver films, there is a strong dependence of the Raman intensity on the oxide thickness. The Raman signal for the optimal SiO(x) interlayer thickness is 38 times higher than the intensity obtained when the Ag particles are deposited directly onto Si (with native oxide). To account for the trends observed in the Raman intensity versus thickness data, calculations of the relative mean square electric field (MSEF) at the surface of the SiO(x) are carried out. These calculations are also used to further optimize the experimental setup.

Hyperspectral confocal fluorescence imaging: Exploring alternative multivariate curve resolution approaches

Abstract: Hyperspectral confocal fluorescence microscopy, when combined with multivariate curve resolution (MCR), provides a powerful new tool for improved quantitative imaging of multi-fluorophore samples. Generally, fully non-negatively constrained models are used in the constrained alternating least squares MCR analyses of hyperspectral images since real emission components are expected to have non-negative pure emission spectra and concentrations. However, in this paper, we demonstrate four separate cases in which partially constrained models are preferred over the fully constrained MCR models. These partially constrained MCR models can sometimes be preferred when system artifacts are present in the data or where small perturbations of the major emission components are present due to environmental effects or small geometric changes in the fluorescing species. Here we demonstrate that in the cases of hyperspectral images obtained from multicomponent spherical beads, autofluorescence from fixed lung epithelial cells, fluorescence of quantum dots in aqueous solutions, and images of mercurochrome-stained endosperm portions of a wild-type corn seed, these alternative, partially constrained MCR analyses provide improved interpretability of the MCR solutions. Often the system artifacts or environmental effects are more readily described as first and/or second derivatives of the main emission components in these alternative MCR solutions since they indicate spectral shifts and/or spectral broadening or narrowing of the emission bands, respectively. Thus, this paper serves to demonstrate the need to test alternative partially constrained models when analyzing hyperspectral images with MCR methods.

Design of a laser-induced breakdown spectroscopy system for on-line quality analysis of pulverized coal in power plants.

Abstract: It is vitally important for a power plant to determine the chemical composition of coal prior to combustion in order to obtain optimal boiler control. In this work, a fully software-controlled laser-induced breakdown spectroscopy (LIBS) system comprising a LIBS apparatus and sampling equipment has been designed for possible application to power plants for on-line quality analysis of pulverized coal. Special attention was given to the LIBS system, the data processing methods (especially the normalization with Bode Rule/DC Level) and the specific settings (the software-controlled triggering source, high-pressure gas cleaning device, sample-preparation module, sampling module, etc.), which gave the best direct measurement for C, H, Si, Na, Mg, Fe, Al, and Ti with measurement errors less than 10% for pulverized coal. Therefore, the apparatus is accurate enough to be applied to industries for on-line monitoring of pulverized coal. The method of proximate analysis was also introduced and the experimental error of Aad (Ash, 'ad' is an abbreviation for 'air dried') was shown in the range of 2.29 to 13.47%. The programmable logic controller (PLC) controlled on-line coal sampling equipment, which is designed based upon aerodynamics, and is capable of performing multipoint sampling and sample-preparation operation.

Perchlorate detection at nanomolar concentrations by surface-enhanced Raman scattering.

Abstract: Perchlorate (ClO4−) has emerged as a widespread environmental contaminant and has been detected in various food products and even in human breast milk and urine. This research developed a sensing technique based on surface-enhanced Raman scattering (SERS) for rapid screening and monitoring of this contaminant in groundwater and surface water. The technique was found to be capable of detecting ClO4− at concentrations as low as 10−9 M (or ∼0.1 μg/L) by using 2-dimethylaminoethanethiol (DMAE) modified gold nanoparticles as a SERS substrate. Quantitative analysis of ClO4− was validated with good reproducibility by using both simulated and contaminated groundwater samples. When coupled with a portable Raman spectrometer, this technique has the potential to be used as an in situ, rapid screening tool for perchlorate in the environment.

Raman chemical imaging of explosive-contaminated fingerprints.

Abstract: Raman chemical imaging (RCI) has been used to detect and identify explosives in contaminated fingerprints Bright-field imaging is used to identify regions of interest within a fingerprint, which can then be examined to determine their chemical composition using RCI and fluorescence imaging Results are presented where explosives in contaminated fingerprints are identified and their spatial distributions are obtained Identification of explosives is obtained using Pearson's cosine cross-correlation technique using the characteristic region (500–1850 cm−1) of the spectrum This study shows the ability to identify explosives nondestructively so that the fingerprint remains intact for further biometric analysis Prospects for forensic examination of contaminated fingerprints are discussed

Self-Weighted Correlation Coefficients and Their Application to Measure Spectral Similarity

Abstract: A technique for spectral searching with noisy data is described that improves the performance over contemporary approaches. Instead of simply calculating the correlation coefficient between the spectrum of an unknown and a series of reference spectra, greater weight is given to the more intense features in the reference spectra. The weight array, w, is given by |r|/{1 + d}, where the vector r represents the reference spectrum and the difference vector, d, contains the difference between the sample and reference data points, equal to |s − kr|, where k is a scaling factor that eliminates the effect of signal strength. By this approach, a large weight is only given to those points that have relatively high absorbance and are close to their counterparts in the reference spectrum. This technique was shown to give significantly improved performance when applied to noisy spectra of trace atmospheric components obtained by target factor analysis.

Potential of Raman Spectroscopy and Imaging Methods for Rapid and Routine Screening of the Presence of Melamine in Animal Feed and Foods

Abstract: The impact of melamine-contaminated animal feed ingredients on food safety has become a major public concern ever since melamine was identified as the organic compound responsible for the deaths of a significant number of cats and dogs in 2007 by way of adulterated pet food. Melamine, a common industrial chemical often added to resins to improve flame resistance and proposed as an alternative form of fertilizer-N for plant growth, was found to be intentionally added to animal feed in amounts ranging from 0.2% to 8% of total mass as a way to boost the products’ apparent protein content. It was also used as a binder in the production of pelleted feed for animals. In addition to melamine, a small amount of cyanuric acid, ammeline, and ammelide were also detected in pet feed and in the tissues and urine of dead pets that had consumed the contaminated food. Even though it is possible that cyanuric acid, ammeline, and ammelide were added, their presence more likely resulted from the degraded derivatives of melamine. There is a great concern that melamine will again enter the food chain and be consumed by humans and animals. As part of the Food Protection Plan, US federal agencies such as the FDA and FSIS and other organizations have established GCMS and LC-MS/MS procedures for the analysis of melamine in food/feed commodities. Although they can detect melamine contaminants in trace amount, these time-consuming laboratory procedures require chemical solvents for the extraction steps and depend on expensive mass spectrometry. Rapid, nondestructive, and routine methods for the specific detection of melamine in raw feed materials are increasingly important, not only for public health concerns but also for melamine screening to prevent protein fraud. Undoubtedly, the well-defined mass spectroscopic technique is preferred due to its low detection limit and capability for structural elucidation; however, since adulteration of raw materials by melamine usually occurs in higher concentrations in order to affect protein content, the high sensitivity of the mass spectroscopic technique may not be necessary. In addition, mass spectrometry might not be sufficiently rapid to screen for the presence of melamine in a large number of food/feed materials from very different sources, because the identification process includes sample-specific extraction procedures, which are labor-intensive and time-consuming. Fast melamine screening requires minimal sample preparation (e.g., no extraction or centrifugation), routine analysis of a number of samples without reagents, minimal procedural steps, and easy operation and interpretation of results. The Raman technique, which has been used to obtain structural information on melamine, is an alternative approach that can be applied to solid materials with no sample pretreatment. In addition, the use of the Fourier transform (FT) methodology and a 1064 nm excitation laser in the near-infrared (NIR) region provides precise wavenumber measurement and good-quality Raman spectra by reducing the interference from fluorescence and photodecomposition of colorants present in food and feed. Raman studies of melamine and melamine-modified resins have been reported in the literature, and the results have revealed the feasibility of the Raman technique for the structural characterization of melamine state in resins. However, there have been few reports on Raman investigation and identification of melamine in complex food and feed systems. The objectives of this study were (1) to identify the characteristic Raman bands in melamine-contaminated wheat flour, corn gluten, and soybean meal mixtures; and (2) to develop simple and universal ratio algorithms for qualitative and quantitative analysis of melamine in mixtures. The ultimate goal is to develop both Raman spectroscopy and Raman chemical imaging methods for rapid, accurate, nondestructive, specific, and routine screening of the presence of melamine in food and feed for public/animal safety and security.

Demountable Liquid/Flow Cell for in Vivo Infrared Microspectroscopy of Biological Specimens

Abstract: We have developed a liquid/flow cell/chamber allowing infrared measurements of living biological specimens with high spatial resolution under a controlled aqueous environment. This flow chamber features sub-micrometer thick diamond windows exhibiting low spherical and chromatic aberrations. Diamond has excellent transmission properties and minimal dispersion over the entire mid-infrared and visible spectral ranges. In contrast to current commercially available infrared liquid chambers, the flow chamber has a slim profile, which accommodates high resolution/magnification microscope objectives with small working distances, down to 0.6 mm above the chamber and 6 mm below the flow chamber. We have coupled a pump to the flow chamber to provide medium exchange. As an example, we present microspectroscopic infrared maps and spectra of the freshwater green alga Micrasterias sp. in the new flow chamber and compare them to maps obtained with a conventional liquid chamber. Pulse-amplitude-modulated fluorescence measurements on Micrasterias sp. cells inside the new flow chamber have been evaluated to demonstrate the viability of the algal cells.

Differentiation of healthy brain tissue and tumors using surface-enhanced Raman scattering.

Abstract: Surface-enhanced Raman scattering (SERS) is a powerful technique for characterization of biological samples. SERS spectra from healthy brain tissue and tumors are obtained by sudden freezing of tissue in liquid nitrogen and crashing and mixing it with a concentrated silver colloidal suspension. The acquired spectra from tissues show significant spectral differences that can be used to identify whether it is from a healthy region or tumor. The most significant change on SERS spectra from the healthy/peripheral brain tissue to tumor is the increase of the ratio of the peaks at around 723 to 655 cm−1. In addition, the spectral changes indicate that the protein content in tumors increases compared to the peripheral/healthy tissue as observed with tumor invasion. The preliminary results show that SERS spectra can be used for a quick diagnosis due to the simplicity of the sample preparation and the speed of the spectral acquisition.

Probing Water and Biomolecules at the Air–Water Interface with a Broad Bandwidth Vibrational Sum Frequency Generation Spectrometer from 3800 to 900 cm −1

Abstract: We have built a broad bandwidth vibrational sum frequency generation (VSFG) spectrometer that can provide high-quality spectra over the range of 3800 to 900 cm−1. The spectrometer contains a commercial Ti:sapphire based 6 W regenerative amplifier as the master light source, a home-built pulse shaper to produce a narrow bandwidth 800 nm beam, a commercial optical parametric amplifier to generate a broad bandwidth femtosecond infrared (IR) pulse, and a detection system with a monochromator and a charge-coupled device (CCD). We applied this spectrometer to obtain VSFG spectra of a lipid monolayer at the air–water interface in the O–H stretching region (3800–3000 cm−1), the C–H stretching region (3100–2700 cm−1), the C–D stretching region (2300–2000 cm−1), the C=O stretching region (1800–1700 cm−1), and the PO2− symmetric stretching region (1200–1000 cm−1). We also obtained the VSFG spectrum of neat water in the O–H stretching region (3800–3000 cm−1) and the VSFG spectrum of a protein, α-synuclein, in the amide I region (1700–1600 cm−1) at the air–water interface. The spectrometer can provide a VSFG spectrum in the O–H stretching region (3800–3000 cm−1) without scanning the IR frequency. This feature will be useful in probing water dynamics at interfaces because the free OH and H-bonded OH can be investigated simultaneously. We have also provided instrumental details and discussed further improvements that should be beneficial to other researchers interested in setting up VSFG instrumentation.

Fourier transform spectrometry with a near-infrared supercontinuum source.

Abstract: Optical fiber based supercontinuum light sources combine the brightness of lasers with the broad bandwidth of incandescent lamps and thus are promising candidates for sources in spectroscopic applications requiring high brightness and broad bandwidth. Herein, near-infrared (IR) Fourier transform (FT) spectrometry with a supercontinuum (SC) light source is investigated. The efficient, collimated propagation of broad bandwidth SC light through an 18 m path length multipass cell is demonstrated. A normalized spectral difference is calculated for the SC spectrum on consecutive FT mirror scans and is found to vary by less than 0.5%, indicating excellent spectral stability. The rms noise on zero absorbance lines is obtained as a function of the number of mirror scans at 0.125, 2, and 16 cm−1 resolution for both the SC and conventional tungsten lamp source. The SC source has approximately a factor of ten times more noise than the lamp under comparable conditions for each resolution and data acquisition time. This clearly indicates that spectral acquisition with the SC source is not detector noise limited. NIR-FT spectra of methane and methyl salicylate, acquired with both the SC and lamp source, are reported. These spectra illustrate the advantage the SC source has over the incandescent source in that it can efficiently traverse long path lengths, thus providing a sensitivity advantage. The spectra also demonstrate the disadvantage of the SC source with respect to the lamp in the increased level of amplitude noise. Prospects for the future use of SC sources in absorption spectroscopy, including possible noise mitigation strategies, are briefly discussed.

Chemical fingerprinting of ready-mixed house paints of relevance to artistic production in the first half of the twentieth century. Part I: Inorganic and organic pigments.

Abstract: This study reports the multi-analytical investigation of ready-mixed house paints used by artists such as Pablo Picasso (1881-1973) in the first half of the twentieth century. The pigment composition of paint swatches on four historic paint sample cards from the Art Institute of Chicago reference collection was characterized by thorough screening using Fourier transform infrared (FT-IR) and X-ray fluorescence (XRF) spectroscopies, followed by Raman spectroscopy when necessary. Spectroscopic investigations highlighted the dominance of zinc-based whites, the consistent choice of particular pigments or their mixtures, as well as the avoidance of others to achieve the various hues on the sample cards. Notable findings included the documentation of strong spectroscopic signatures of metal soaps. Given the similarities in composition of early twentieth century artists' and house paints, the results indicate that the identification of house paints in works by Pablo Picasso and others must be based on a combination of parameters rather than the detection of a single chemical marker. Results have been applied to the case study of Picasso's 1935 sculpture Figure (AIC 1988.428), which incorporates direct evidence of the use of house paint by the artist.

Digital Dewaxing of Raman Signals: Discrimination Between Nevi and Melanoma Spectra Obtained from Paraffin-Embedded Skin Biopsies

Abstract: Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

An insight into sequential order in two-dimensional correlation spectroscopy.

Abstract: Determination of the sequential order of events is a very important feature of generalized two-dimensional correlation spectroscopy (2D-COS). Recently, queries have been put forward on the actual effectiveness of this method when changes are non-periodic, particularly in the presence of local sequential order. Consequently, it brings an urgent necessity to understand the true connotation of the sequential order parameter derived from 2D-COS analysis. This article presents an attempt to address these questions based on the analysis of simulated spectra by assuming the band intensity changes in a logarithmic, exponential, hyperbolic, or polynomial manner. It is concluded that for two events changing monotonically and without local sequential orders, one event occurring prior to the other as determined using 2D-COS means in most cases that the former has both a shorter half-time and a greater half-intensity. As a rule of thumb, intensity versus the perturbation factor should be plotted before performing the 2D-COS analysis to determine the sequential order of the involved events. In the presence of obvious local sequential order, 2D-COS analysis is unnecessary. Otherwise, sequential order can be determined quite reliably based on 2D-COS analysis.

Multiple Perturbation Two-Dimensional Correlation Analysis of Cellulose by Attenuated Total Reflection Infrared Spectroscopy

Abstract: An extension of the two-dimensional (2D) correlation analysis scheme for multi-dimensional perturbation is described. A simple computational form is provided to construct synchronous correlation and disrelation maps for the analysis of microscopic imaging data based on two independent perturbation variables. Sets of time-dependent attenuated total reflection infrared (ATR-IR) spectra of water and cellulose mixtures were collected during the evaporation of water from finely ground cellulose. The system exhibits complex behaviors in response to two independent perturbations, i.e., evaporation time and grinding time. Multiple perturbation 2D analysis reveals a specific difference in the rate of evaporation of water molecules when accompanied by crystallinity changes of cellulose. It identifies subtle differences in the volatility of water, which is related to the crystalline structure of cellulose.