Fluorescence lifetime imaging and Fourier transform infrared spectroscopy of Michelangelo's David

We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.     

Classification of Fourier transform infrared microscopic imaging data of human breast cells by cluster analysis and artificial neural networks

Cluster analysis and artificial neural networks (ANNs) are applied to the automated assessment of disease state in Fourier transform infrared microscopic imaging measurements of normal and carcinomatous immortalized human breast cell lines. K-means clustering is used to implement an automated algorithm for the assignment of pixels in the image to cell and non-cell categories. Cell pixels are subsequently classified into carcinoma and normal categories through the use of a feed-forward ANN computed with the Broyden-Fletcher-Goldfarb-Shanno training algorithm. Inputs to the ANN consist of principal component scores computed from Fourier filtered absorbance data. A grid search optimization procedure is used to identify the optimal network architecture and filter frequency response. Data from three images corresponding to normal cells, carcinoma cells, and a mixture of normal and carcinoma cells are used to build and test the classification methodology. A successful classifier is developed through this work, although differences in the spectral backgrounds between the three images are observed to complicate the classification problem. The robustness of the final classifier is improved through the use of a rejection threshold procedure to prevent classification of outlying pixels.     

Fourier transform infrared spectral analysis of degenerative cartilage: an infrared fiber optic probe and imaging study

A preliminary investigation into the diagnostic potential of an infrared fiber optic probe (IFOP) for evaluating degenerative human articular cartilage is described. Twelve arthritic human tibial plateaus obtained during arthroplasty were analyzed using the IFOP. Infrared spectra were obtained from IFOP contact with articular surface sites visually graded normal or degraded (Collins Scale grade 1 and grade 3, respectively). Comparisons of infrared spectral parameters (peak heights and areas) were made to elucidate spectral indicators of surface degeneration. IFOP spectral analysis revealed subtle but consistent changes between grades 1 and 3 sites. Infrared absorbance bands arising from type II collagen were observed to change with degradation. More degraded tissues exhibited increased amide II (1590-1480 cm(-1))/1338 cm(-1) area ratio (p=0.034) and decreased 1238/1227 cm(-1) peak ratio (p = 0.017); similar changes were seen with Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Grades 1 and 3 cartilage showed consistent spectral differences in the amide II, III, and 1338 cm(-1) regions that are likely related to type II collagen degradation that accompanies cartilage degeneration. These results suggest that it may be possible to monitor subtle changes related to early cartilage degeneration, allowing for IFOP use during arthroscopy for in situ determination of cartilage integrity.     

Gram-Schmidt orthogonalization for rapid reconstructions of Fourier transform infrared spectroscopic imaging data

Increasingly voluminous Fourier transform infrared (FT-IR) spectroscopic imaging data sets are being generated with the advent of both faster array detectors and the implementation of time-resolved imaging techniques, resulting in data processing becoming the limiting step in visualizing sample heterogeneity and temporal profile evolution. We report the application of a Gram-Schmidt vector orthogonalization procedure in interferogram space to provide a significant time saving advantage in processing of one to two orders of magnitude in comparison to conventional spectral processing. Illustrative data from human skin biopsies and from dynamic molecular reorganizations within liquid crystalline microdomains is employed to discuss the capabilities and limitations of this information-extraction approach.     

Fourier transform infrared imaging of stereoregular poly(methyl methacrylate) dissolution

Fourier transform infrared (FT-IR) imaging was used to study dissolution of stereoregular poly(methyl methacrylate) (PMMA) films in toluene, benzene, chloroform, acetic acid, and 2-ethoxyethanol. Images of the polymer-solvent interface showed that syndiotactic (sPMMA), atactic (aPMMA), and isotactic (iPMMA) samples dissolved in acetic acid and chloroform; only iPMMA dissolved in benzene, toluene, and 2-ethoxyethanol. Concentration profiles allowed quantitative comparison of each polymer-solvent system. In the cases of chloroform and acetic acid, the rate of polymer dissolution increased with increasing isotacticity. Dissolution of PMMA in chloroform was observed to coincide with development of strong polymer-solvent interactions. The results of systems containing toluene, benzene, and 2-ethoxyethanol reflect kinetic effects on dissolution, including solvent size and stiffness of the polymer backbone.     

Combining the tape-lift method and Fourier transform infrared spectroscopic imaging for forensic applications

Conventional Fourier transform infrared (FT-IR) spectroscopy and microscopy have been widely used in forensic science. New opportunities exist to obtain chemical images and to enhance the spatial resolution using attenuated total reflection (ATR) FT-IR spectroscopy coupled with a focal-plane array (FPA) detector. In this paper, the sensitivity limits of FT-IR imaging using three different ATR crystals (Ge, ZnSe, and diamond) in three different optical arrangements for the detection of model particles is discussed. Model systems of ibuprofen and paracetamol particles having sizes below 32 mum were studied. The collection of drug particles was achieved with the aid of two different tapes: common adhesive tape and a film of polydimethylsiloxane (PDMS). The surface of the film with collected particles was measured directly via ATR-FT-IR imaging. Since the removal of tape from porous surfaces can be difficult, the application of micro ATR-FT-IR imaging directly to the surface of a newspaper contaminated with particles of model drugs is also discussed. In order to assess the feasibility of the chosen method in a forensic case study, the detection of diacetylmorphine hydrochloride traces in PDMS matrix and the finger surface is investigated. The scenarios considered were that of the detection of evidence collected at a crime scene with the tape lift method and the analysis of the finger of an individual after drug handling. The results show broad implications in the detection of drugs of abuse.     

Fourier transform spectroscopy of LaS in the infrared

We recorded the emission spectrum of diatomic lanthanum sulfide on the Los Alamos Fourier transform spectrometer. In the region 7500-16,000 cm(-1), we identified over 120 bands and assigned them to the A(2)∏(r)-X(2)Σ(+) and B(2)Σ(+)-X(2)Σ(+) transitions. Each of these bands is four headed.     

Modeling and quantifying biochemical changes in C6 tumor gliomas by Fourier transform infrared imaging

The purpose of the study was to investigate molecular changes associated with glioma tissues using FT-IR microspectroscopic imaging (FT-IRM). A multivariate statistical analysis allowed one to successfully discriminate between normal, tumoral, peri-tumoral, and necrotic tissue structures. Structural changes were mainly related to qualitative and quantitative changes in lipid content, proteins, and nucleic acids that can be used as spectroscopic markers for this pathology. We have developed a spectroscopic model of glioma to quantify these chemical changes. The model constructed includes individual FT-IR spectra of normal and glioma brain constituents such as lipids, DNA, and proteins (measured on delipidized tissue). Modeling of FT-IR spectra yielded fit coefficients reflecting the chemical changes associated with a tumor. Our results demonstrate the ability of FT-IRM to assess the importance and distribution of each individual constituent and its variation in normal brain structures as well as in the different pathological states of glioma. We demonstrated that (i) cholesterol and phosphatidylethanolamine contributions are highest in corpus callosum and anterior commissure but decrease gradually towards the cortex surface as well as in the tumor, (ii) phosphatidylcholine contribution is highest in the cortex and decreases in the tumor, (iii) galactocerebroside is localized only in white, but not in gray matter, and decreases in the vital tumor region while the necrosis area shows a higher concentration of this cerebroside, (iv) DNA and oleic acid increase in the tumor as compared to gray matter. This approach could, in the future, contribute to enhance diagnostic accuracy, improve the grading, prognosis, and play a vital role in therapeutic strategy and monitoring.     

Macromolecular concentrations in bovine nasal cartilage by Fourier transform infrared imaging and principal component regression

Fourier transform infrared imaging (FT-IRI) and principal component regression (PCR) were used to quantitatively determine collagen and proteoglycan concentrations in bovine nasal cartilage (BNC). An infrared spectral library was first established by obtaining eleven infrared spectra from a series of collagen and chondroitin 6-sulfate mixed in different ratios. FT-IR images were obtained from 6-μm-thick sections of BNC specimens at 6.25-μm pixel size. The spectra from the FT-IR images were imported into a PCR program to obtain the relative concentrations of collagen and proteoglycan in BNC, based on the spectral library of pure chemicals. These PCR-determined concentrations agreed with the molecular concentrations determined biochemically using an enzyme digestion assay. Use of the imaging approach revealed that proteoglycan loss in the specimens occurs first at the surface of the tissue block when compared with the middle portion of the tissue block. The quantitative correlation of collagen and proteoglycan revealed that their infrared absorption peak areas at 1338 and 1072-855 cm(-1) can only be used as qualitative indicators of the molecular contents. The use of PCR with FT-IRI offers an accurate tool to spatially determine the distributions of macromolecular concentration in cartilage.     

Application of Fourier transform infrared spectroscopic imaging to the study of effects of age and dietary l-arginine on aortic lesion composition in cholesterol-fed rabbits

Diet-induced atherosclerotic lesions in the descending thoracic segment of rabbit aorta were analysed ex vivo by micro-attenuated total reflection (ATR)–Fourier transform infrared (FTIR) spectroscopic imaging. The distribution and chemical character of lipid deposits within the arterial wall near intercostal branch ostia were assessed in histological sections from immature and mature rabbits fed cholesterol with or without l-arginine supplements. Previous studies have shown that both these properties change with age in cholesterol-fed rabbits, putatively owing to changes in the synthesis of nitric oxide (NO) from l-arginine. Immature animals developed lesions at the downstream margin of the branch ostium, whereas lipid deposition was observed at the lateral margins in mature animals. Dietary l-arginine supplements had beneficial effects in mature rabbit aorta, with overall disappearance of the plaques; on the other hand, they caused only a slight decrease of the lipid load in lesions at the downstream margin of the ostium in immature rabbits. ATR–FTIR imaging enabled differences in the lipid to protein density ratio of atherosclerotic lesions caused by age and diet to be visualized. Lipid deposits in immature rabbits showed higher relative absorbance values of their characteristic spectral bands compared with those in immature l-arginine-fed rabbits and mature rabbits. The multivariate methods of principal component analysis (PCA) and factor analysis (FA) were employed, and relevant chemical and structural information were obtained. Two distinct protein constituents of the intima–media layer at different locations of the wall were identified using the method of FA. This approach provides a valuable means of investigating the structure and chemistry of complex heterogeneous systems. It has potential for in vivo diagnosis of pathology.     

Studies of poly(styrene-co-allyl alcohol)/polyester blends using Fourier transform infrared spectroscopy and imaging

The interaction of poly(styrene-co-allyl alcohol) and aliphatic polyester was studied using FT-IR spectroscopy. The hydrogen bonding between two polymers was investigated by monitoring C=O and OH stretching modes with the variation of concentration and temperature. The phase-separated morphology was also observed using FT-IR imaging, where the spectroscopic analysis of each phase is available.     

Reference sample method reduces the error caused by variable cryosection thickness in Fourier transform infrared imaging

Fourier transform infrared imaging (FT-IRI) is a novel technique for characterization of the biochemical composition of biological tissues, e.g., articular cartilage. The use of cryosections is preferred in FT-IRI. Unfortunately, significant variation in section thickness often impairs the suitability of cryosections for quantitative FT-IRI analysis. The present study introduces an inexpensive reference sample method for quantitative analysis. In this technique, specimen absorption is normalized with that of nitrocellulose membrane embedded and cryosectioned with the sample. Mean variation of the infrared absorption in cartilage specimens was 11.5%, 12.1%, and 20.6% for 5 microm, 10 microm, and 14 microm thick sections, respectively, without normalization. Normalization reduced the variation to 5.2%, 4.0%, and 4.6% for the same sections, respectively. The normalization method enables usage of cryosections for quantitative work and significantly reduces the cost and time needed for FT-IRI analysis.     

Application of Fourier transform infrared spectroscopy in cement Alkali quantification

Alkali in cement is responsible for the Alkali–silica-reaction phenomenon that manifests itself in the form of premature cracking in concrete structures such as bridge decks and concrete pavements. X-ray fluorescence spectroscopy (XRF) is commonly used for cement Alkali quantification but a simpler and faster analytical procedure based on Fourier transform infrared spectroscopy (FTIR) has been expanded for this purpose. An analytical absorption band at 750 cm^?1 in the FTIR spectra of cement samples belonging to Alkali solid solution of tricalcium aluminate [C₃A(ss)] is used for Alkali quantification. Regression analysis of a plot correlating FTIR absorption band area ratio (750/923 cm^?1) to equivalent Alkali Na₂O _e (Na₂O _e  = % Na₂O + 0.658 × % K₂O) measured by XRF shows a linear correlation coefficient, R ², of 0.97. High Alkali cement samples show a higher microstructural disorder coefficient, C _d, which is a reactivity criterion introduced by Bachiorrini and co-authors (Proceedings of the seventh international conference on concrete alkali-aggregate reactions? 1986) for ASR-susceptible aggregates. Results of this research indicate applicability of FTIR technique to quantitatively predict cement vulnerability to ASR through the \( A_{{750\,{\text{cm}}^{ - 1} }} \) to \( A_{{923\,{\text{cm}}^{ - 1} }} \) band area ratio and the magnitude of the disorder coefficient (C _d).     

Fourier transform infrared spectroscopy of size-segregated aerosol deposits on foil substrates

A method based on Fourier transform infrared (FTIR) double-pass transmittance spectroscopy was developed for determining functional group loading in size-segregated ambient aerosol deposits. The impactor employed for sample collection utilized rotating stages, which produced uniform particulate matter (PM) deposits on standard Al foil substrates. Each sample was analyzed without extraction using an FTIR spectrometer equipped with a reflectometer accessory. The use of the reflectometer obviated the need for infrared window materials as substrates. (NH(4))(2)SO(4) aerosol generated under laboratory conditions were used to calibrate deposit mass to the band strength of the relatively isolated nu(4) bending mode of SO(2-)(4) centered near 620 cm(-1). Atmospheric PM was sampled during the summer of 2004 in Huntsville, Ala. Sulfate concentrations determined in this initial study correlated well with measurements made by collocated EPA air samplers.     

Fourier transform infrared spectroscopic imaging of anisotropic poly(vinylidene fluoride) films with polarized radiation

The technique of Fourier transform infrared (FT-IR) spectroscopic imaging with focal plane array detectors has proved to be a powerful technique for rapid chemical visualization of samples with a lateral resolution up to about 10 mum. However, the potential of FT-IR imaging for the characterization of anisotropic materials can be significantly enhanced by using polarized radiation. This issue will be addressed in the present communication, which reports for the first time imaging investigations based on the FT-IR polarization spectra of poly(vinylidene fluoride) films that have been uniaxially elongated below and above the threshold temperature of the II(alpha) --> I(beta) phase transition.     

Attenuated total reflection Fourier transform infrared imaging with variable angles of incidence: a three-dimensional profiling of heterogeneous materials

Depth profiling in Fourier transform infrared (FT-IR) spectroscopic imaging has been demonstrated using a single reflection variable angle attenuated total reflection (ATR) accessory. Chemical information about samples can be obtained in three dimensions by acquiring ATR-FT-IR images at different angles of incidence through the ATR crystal. The image quality and field of view achieved at different angles of incidence has been discussed. A polymer film comprising two layers has been used as an example to demonstrate the principle of the measurement. The demonstrated approach is a promising tool to obtain depth profiles of heterogeneous materials. The extent of the measured depths is limited and ranges from approximately 0.3 to 4 microm, but the spatial resolution in the z-direction is not limited by diffraction. The development of this approach opens up the possibility to study the spatial heterogeneity of thin films including biological tissues, such as hair and skin, with high depth resolution.     

Quantitative determination of moisture in lubricants by Fourier transform infrared spectroscopy

This paper describes the development of a practical Fourier transform infrared (FT-IR) method for the determination of moisture in lubricants through the combined use of signal transduction and differential spectroscopy to circumvent matrix effects. The acid-catalyzed stoichiometric reaction of 2,2-dimethoxypropane (DMP) with moisture to produce acetone was used to provide IR signals proportional to the amount of moisture present in oils. Calibration standards were prepared by spiking polyalphaolefin (PAO) gravimetrically with water using dioxane as a carrier. For FT-IR analysis, standards and samples were diluted with acidified isooctane and then split, with one aliquot treated with DMP and the other with a blank reagent. The spectra of the two aliquots were collected, and a differential spectrum was obtained so as to ratio out the invariant spectral contributions from the sample. Quantitation for moisture was based on measurement of the peak height of the nu(C=O) absorption of acetone at 1717 cm(-1), yielding a standard error of calibration of approximately 40 ppm H2O. The method was validated by standard addition of water in dioxane to PAO containing added base as well as to new and used oils. In all cases the method responded quantitatively to standard addition, the average standard error of prediction being approximately 80 ppm, with the results showing only a minor dependence on the oil formulation. From an analytical perspective, the FT-IR method is both more reproducible and more accurate than Karl Fischer methods and has advantages in terms of environmental considerations, sample size, and speed of analysis as well as the variety of oil types that can be handled. Signal transduction/differential spectroscopy may have broader utility as an alternative means for the determination of low levels of moisture in complex matrices.     

Fourier transform infrared study of lipoxygenase conformation in organic solvent media

The secondary structure of commercially purified soybean lipoxygenase (EC 1.13.11.12) was investigated in selected monophasic organic solvents, including chloroform, methanol, acetonitrile, hexane, and octane. The Fourier transform infrared (FT-IR) spectra of the enzyme obtained in chloroform, methanol, and acetonitrile showed an absorption band at 1617 cm(-1) indicative of significant protein aggregation, whereas spectra of lipoxygenase in hexane and octane exhibited substantially less aggregate formation. Variable-temperature infrared studies of lipoxygenase in D(2)O show that the predominately alpha-helical structure of the protein undergoes an irreversible transition to intermolecular beta-sheet at and above 65 degrees C. Chemical imaging technology employing an FT-IR spectrometer equipped with an infrared microscope and a focal-plane array detector was used to examine the changes in the secondary structure of lipoxygenase at the water-hexane interface in the presence and absence of substrate. The secondary structure of lipoxygenase at the hexane-water interface was comparable to that of the structure of lipoxygenase in D(2)O after exposure of lipoxygenase solution to hexane.