The influence of cholesterol (CHO) on the phase behavior of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) planar supported lipid bilayers (PSLBs) was investigated by sum-frequency vibrational spectroscopy (SFVS). The intrinsic symmetry constraints of SFVS were exploited to measure the asymmetric distribution of phase segregated phospholipid domains in the proximal and distal layers of DSPC + CHO binary mixtures as a function of CHO content and temperature. The SFVS results suggest that cholesterol significantly affects the phase segregation and domain distribution in PSLBs of DSPC in a concentration dependent manner, similar to that found in bulk suspensions. The SFVS spectroscopic measurements of phase segregation and structure change in the binary mixture indicate that membrane asymmetry must be present in order for the changes in SFVS signal to be observed. These results therefore provide important evidence for the delocalization and segregation of different phase domain structures in PSLBs due to the interaction of cholesterol and phospholipids.
Fourier transform infrared microscopic imaging (FTIRI) was used to quantitatively examine the anisotropies of proteoglycan (PG) and collagen in articular cartilage. Dried 6 μm thick sections of canine humeral cartilage were imaged at 6.25 μm pixel-size in FTIRI with an infrared analyzer set at 26 different angles between 0° and 180° polarization. Like the amide II and amide III peaks, the 1338 cm(-1) band confirms the anisotropy of collagen fibrils in cartilage. The absorption profile of the sugar band shows an anisotropic flipping at the deeper part in the radial zone, just above the tidemark. Together with the reduction in the PG concentration and subsequent increase in tissue calcification in this region, this anisotropy flipping of sugar might be caused by the orientational change in the collagen-attaching PG from orthogonal to parallel when the fibrils are entering the calcified zone.
The integration of near IR picosecond pulse excitation, collinear beam geometry, epi-detection, and laser-scanning has produced a coherent anti-Stokes Raman scattering (CARS) microscope with a detection sensitivity of 10(5) vibrational oscillators, sub-micron 3D resolution, and video-rate acquisition speed. The incorporation of spectral detection and other imaging modalities has added versatility to the CARS microscope. These advances allowed sensitive interrogation of biological samples, particularly lipids that have a high density of CH(2) groups. With initial applications to membrane domains, lipid bodies, demyelinating diseases, obesity, and cardiovascular diseases, CARS microscopy is poised to become a powerful bio-imaging tool with the availability of a multifunctional, affordable, easy-to-operate CARS microscope, and the development of CARS endoscopy for in vivo diagnosis.
In this contribution, we discuss state-of-the-art methodology for the collection and analysis of hyperspectral images of tissue that will become useful in complementing classical histopathology. In particular, we discuss sampling strategies, data collection methods, and computational approaches to produce pseudo-color maps of large tissue sections of lymph nodes, up to about 100 mm(2) in size. The latter efforts include methods to reduce the presence of dispersion artifacts in IR transflection micro-spectra which can greatly impact the statistical analyzes performed on the data, such as hierarchical cluster analysis and principal components analysis.
Histopathology forms the gold standard for the diagnosis of breast cancer. Fourier Transform Infrared (FT-IR) spectroscopic imaging has been proposed to be a potentially powerful adjunct to current histopathological techniques. Most studies using FT-IR imaging for breast tissue analysis have been in the transmission or transmission-reflection mode, in which the wavelength and optics limit the data to a relatively coarse spatial resolution (typically, coarser than 5 μm × 5 μm per pixel). This resolution is insufficient to examine many histologic structures. Attenuated Total Reflectance (ATR) FT-IR imaging incorporating a Germanium optic can allow for a four-fold increase in spatial resolution due to the material's high refractive index in the mid-IR. Here, we employ ATR FT-IR imaging towards examining cellular and tissue structures that constitute and important component of breast cancer diagnosis. In particular, we resolve and chemically characterize endothelial cells, myoepithelial cells and terminal ductal lobular units. Further extending the ability of IR imaging to examine sub-cellular structures, we report the extraction of intact chromosomes from a breast cancer cells and their spatially localized analysis as a novel approach to understand changes associated with the molecular structure of DNA in breast cancer.
We report microscopically collected infrared spectra of cells found in human urine in an effort to develop automatic methods for bladder cancer screening. Unsupervised multivariate analysis of the observed spectral patterns reveals distinct spectral classes, which correlated very well with visual cytology. Therefore, we believe that spectral analysis of individual cells can aid cytology in rendering reliable diagnoses based on objective measurements and discriminant algorithms.
We discuss the causes contributing to the variance of the spectra of individual human epithelial cells. This aspect has largely been ignored in previous studies, but needs to be understood for diagnostic applications of infrared micro-spectroscopy. We attribute the spectral variance to Mie scattering, and to variations of nuclear contributions to the overall spectra caused by different nuclear size.
Vibrational spectroscopy (Infrared and Raman), and in particular micro-spectroscopy and micro-spectroscopic imaging has been used to characterize developmental changes in bone and other mineralized tissues, to monitor these changes in cell cultures, and to detect disease and drug-induced modifications. Examples of the use of infrared micro-spectroscopy and micro-spectroscopic imaging are discussed in this review.
FT-IR spectra of the title compounds and derivatives were measured in the range 4000–120 cm−1. A complete experimental spectral assignment has been performed. A simplified normal coordinate treatment orientates some assignment of frequencies in the low energy region. Bands at ca. 420 and 300 cm−1 are ascribed to the vCu-N modes. A stability order of the Cu(I) complexes is proposed on the basis of their Cu-N frequency values.
Equilibrium geometries, rotational constants, harmonic vibrational frequencies, infrared intensities, Raman activities, and 1H and 13C NMR spectra were calculated for 1,2-dimethylenecyclobutane and its less stable isomer 1,3-dimethylenecyclobutane by using MP2, DFT (B3PW91), and RHF theoretical methods involving the 6-311++G∗∗ basis set.The properties calculated theoretically have been compared with the experimental values. The internal coordinates defined for both isomers were used in the potential energy distribution (PED) analysis. The theoretical vibrational and NMR spectra form the basis to differentiate particular compounds in reaction mixture.
The conformational equilibrium of 1,2-dibromoethane (DBE) in various media (vapor phase, liquid, and solutions in n-hexane, carbon tetrachloride, toluene, carbon disulfide, bromoform, acetone, nitromethane, deuterated acetonitrile and deuterated dimethylsulfoxide) has been studied by IR absorption spectra. The enthalpy differences between trans (t) and gauche (g) conformers (ΔHo) were determined from the dependencies of ln(It/Ig) upon T−1, where It and Ig are the integrated intensities of the bands, belonging to different conformers. The values RT·ln(It/Ig) and (R·ln(It/Ig)+ΔHo/T) obtained at 296 K were used as measures of the free enthalpy (ΔGo) and entropy differences of the conformers (ΔSo) respectively, when considering their changes with solvent. To minimize the errors due to solvent and temperature effects on the IR-band absorption coefficients, four different band pairs of trans and gauche conformers were investigated. Good correlations between ΔGo, ΔHo and the function of dielectric permitivity of the medium (0.5−(ε−1)/(2ε+1))1/2 were observed for all solvents except toluene. Significant changes of ΔSo with the media have been found: they cover more than 1.5 cal mol−1 K−1 when going from the vapor phase to polar solvents. The ΔSo values correlate with ΔHo ones (compensation effect); the slope of the dependence (ΔΔSo/ΔΔHo)=(1.0±0.3)·10−3 K−1 is close to those determined earlier for 1,2-dichloro- and bromofluoro-ethanes, trans-1,2-dichlorocyclohexane and o-iodophenol. The overall integrated intensities in the CH2-stretching (αA/B(str), 3200–2700, cm−1) and deformational (αA/B(def), 1550–1300, cm−1) regions were measured for neat DBE and its solutions in CCl4, CD3CN and (CD3)2SO. The αA/B(str) value noticeably increases when going from CCl4 to proton acceptor solvents, while the αA/B(def) does not depend on the solvent. These results are interpreted within the framework of the formation of weak hydrogen bonds between CH2-groups of DBE and proton acceptor groups of the solvents. The enthalpies of specific interaction of DBE with CD3CN and (CD3)2SO were estimated using the `intensity rule'. These values are about 1 kcal mol−1. Nevertheless, poor correlation between ΔHo, ΔGo and basicity parameters of the solvents indicate the minor effect of the H-bond formation on the conformational equilibrium of DBE.
Post–Hartree–Fock MP2 and density functional theory (DFT) calculations with the combined B3LYP functional using the 6-31G(d) basis set were carried out to investigate structure and vibrational spectra of 3H-1,2-dithiole-3-thione and 3H-1,2-dithiole-3-one. Theory predicts planar structures of Cs symmetry in both cases. The geometric parameters and the results of the Natural Bond Orbital (NBO) and Natural Resonance Theory (NRT) calculations are compatible with structures with localized rather than delocalized bonds. Schleyer's Nuclear Independent Chemical Shielding (NICS) criterion also indicates relatively low aromatic delocalization. Both the MP2 and DFT calculations provided consistent results about the fundamental vibrational modes and their assignment. Uniformly scaled DFT frequencies reproduce the experimental data in CCl4 and CS2 solutions not as well as uniformly scaled MP2 frequencies. The extension of the standard basis set of DFT calculations from 6-31G(d) to 6-311+G(2df,2p) affects vibrational frequencies and intensities to a minor extent.
The electrosorption of 4-isopropylsulfanylmethyl-1,2,3-triazole (ISMT) on a polycrystalline gold electrode has been studied with cyclic voltammetry and surface enhanced Raman spectroscopy (SERS). Vibrational wavenumbers were calculated for the conceivable tautomers with density function theory (DFT) at the B3LYP level using the 6-31G* basis set. The data obtained from vibrational frequency calculations are used to assign vibrational bands obtained in infrared and Raman spectroscopy as well as in surface enhanced Raman spectroscopy of the molecule adsorbed on a roughened gold electrode surface. Results imply a gold–sulfur interaction with ISMT adsorbed intact (i.e. without C–S bond cleavage) onto the gold surface from neutral solution. Beyond a gold–sulfur stretching mode, this suggestion is supported by significantly enhanced internal modes of the methyl groups in the isopropyl substituent and an almost complete lack of shift of band positions of the triazole ring itself being expected in case of a gold–nitrogen interaction. With an acidic electrolyte solution, a gold–sulfur vibrational mode typical of a thiolate-like adsorbate is found, this indicates dissociative adsorption.
Infrared spectroscopy studies of 1,3-indanedione (ID) in 16 different pure organic solvents were undertaken to investigate the solvent–solute interactions and to correlate solvent properties, such as the Kirkwood–Bauer–Magat (KBM) equation, the solvent acceptor number (AN) and the linear solvation energy relationships (LSER), using the infrared band shift. The correlations were performed both symmetric and asymmetric band stretching. Poor correlation between dielectric constants and the frequencies in both symmetric and asymmetric CO stretching showed that the KBM relationship was unsuitable for the molecule studied in this work. These frequencies exhibited a better correlation with the LSER than the solvent AN. The asymmetric CO stretching was found in better correlation than symmetric.
This paper discusses the nature of the products formed from the reaction of 2-pyridinecarboxaldehyde and methylamine. The synthesized products manifest themselves in two distinct forms, one an oil and the other crystalline, which have identical chemical properties but differ dramatically in their spectroscopic parameters. A novel trimeric structure, the hexahydro-s-triazine, is suggested for the crystalline form, while the oil appears to be the monomeric N-methyl pyridine-2-carboxaldimine. IR, UV–vis, Raman and NMR spectroscopies are used to validate the proposed structures. The conversion of the trimeric crystalline form to the monomeric oil form, is acid-catalyzed and occurs rapidly.
The Fermi resonance interaction between the methylene symmetric CH stretching mode and appropriate binary combinations involving the methylene bending mode in the Raman spectra of polyamines, namely in 1,6-hexanediamine derivatives, under different conditions of deuteration and ionisation of the head amino groups and varying temperature has been analysed. The tentative assignment of the observed bands in the CH stretching region of the registered Raman spectra is reported and the ratio of the Raman intensities of the symmetric stretching to the antisymmetric fundamentals is considered in its possible relation to the order/disorder of the skeletal hydrocarbon chains, from the all-trans prevailing geometry in the solid state at lower temperatures to the increasing amount of gauche arrangements on heating and melting.
Near-infrared (near-IR) and mid-infrared (mid-IR) vibrational circular dichroism (VCD) spectra in the region 800–10,000 cm−1 were recorded for nine enantiomeric pairs (or enantiomer and racemate pairs) of molecules with three ChiralIR Fourier transform (FT) VCD spectrometers equipped with various light sources and detectors and modified for dual polarization modulation (DPM) operation. Correlations across these sets of spectra permit conclusions regarding expected VCD intensities in the near-infrared compared to the conventional mid-IR regions. These VCD spectra also provide spectral standards for future studies in the near-infrared region. Compared with recent near-IR measurements carried out elsewhere using dispersive instrumentation, better signal-to-noise ratios were obtained for all but the frequency region above 8000 cm−1 where VCD spectra of comparable quality were obtained. These results are the first comprehensive set of VCD spectra for a set of simple chiral molecules with continuous coverage from the mid-infrared to nearly the visible region of the spectrum. This study demonstrates that with suitably equipped FT-VCD spectrometers, VCD and IR spectra can be measured with high quality across a wide spectral range.
Infrared spectroscopy measurements on different hemoproteins and models of the active side have been completed for the spectral range from 1800 to 100 cm−1 giving an overview on the contributions expected in the low frequency range. Little is known of the low frequency contribution of proteins in infrared. In order to detect the contributions of heme centers and protein moiety, a systematic study of the infrared spectroscopic properties of the porphyrin ring, the ferric porphyrines with different ligands (hemine and hematine), a heme with 11 amino acids (microperoxidase-11), cytochrome c and cytochrome c oxidase are compared at different pD values and an overview on the relative contributions of hemes, their ligands and the protein site can be provided in the low frequency region. Beside the well know amide I and II modes, the low frequency range is found to be dominated by the amide IV and VI mode around 530/580 cm−1 for cytochrome c and cytochrome c oxidase, as well as further proteins like ferrodoxin. Below 300 cm−1 amide VII modes, doming modes of the heme site and hydrogen-bonding signatures overlap to a broad peak with covering 100–250 cm−1. As clear markers for the iron ligands, bands can be depicted at 388/378 cm−1 (FeN, histidine ligand) and 345 cm−1 FeCl. Furthermore the ring vibration of the protonated histidine is determined at 623 cm−1.
Recent epidemiological studies have positively associated total dietary fat intake to prostate cancer (CaP) incidence and progression. However, the role of fat-specific intake remains unclear through these patient-orientated studies, which in-turn warrants the need for controlled molecular based investigations. FTIR microspectroscopy is now a well established tool for the metabolomic analysis of a wide variety of biomolecules at the whole cell level and can be used to generate hypotheses to refine molecular specific experiments. For the first time, we have used FTIR microspectroscopy to measure the uptake and metabolism of the saturated fatty acid (FA), palmitic acid (PA) and the unsaturated FA, arachidonic acid (AA) using deuterated analogues. We also report on the temporal fluctuations of different biomolecular domains (lipid, phosphate and protein secondary structure) within PC-3 cells in response to D8-AA and D31-PA uptake. Our results demonstrate that (i) FTIR can be used to track eicosonoid generation in D8-AA stimulated cells; (ii) the intracellular management of D31-PA at high-loadings can be elucidated by monitoring lipid biosynthesis through the lipid hydrocarbon peak area signal; (iii) metabolism of lipid pools results in significant protein phosphorylation.
Variable-temperature infrared (IR) spectra of cyclohexane and IR and Raman spectra of chlorocyclohexane have been investigated by graphic eigenvalue analysis. Thermal effects known as peak shift and band broadening combined with heteroscedastic noise in vibrational bands are found to have severe influence on the interpretation of the outcome of rank analysis. Methods for correction of frequency shifts and band broadening in the spectral profiles due to temperature variation are developed and tested.
We report on the coupling of an NIR laser operating at 1064 nm with a dispersive Raman spectrometer using a germanium array detector to measure the Stokes-shifted Raman spectra of explosives. By using a Ge array detector, spectral features from 250 to 1800 cm−1 could clearly be observed for the majority of compounds studied. Spectra were routinely obtained with 1–2 min of data collection. The utility of this instrument is compared to other previous identified instrumental approaches, with particular regard to fieldability of a Raman-based explosive analyzer.
A new infrared (IR) transmission cell has been developed which allows in situ catalyst pretreatment under well-controlled conditions, followed by the characterization with IR spectroscopy on adsorbed CO at 120 K. The temperature of the sample can be controlled accurately from 120 up to 773 K while the sample is present in the IR beam. In an integrated reactor, the sample can be heated from 300 up to 1273 K under reactive conditions, for instance in H2S. To ease the comparison of different samples pretreated under identical conditions, for instance for background corrections, the sample holder may contain two samples. The transmission cell is high vacuum compatible and has been built in a transportable frame allowing a flexible use of the IR spectrometer. The performance of the cell is demonstrated for the characterization of an oxidic Ni/Al2O3 sample and a reduced WO3/Al2O3 sample.
As a molecular probe of tissue composition, infrared spectroscopic imaging can potentially serve as an adjunct to histopathology in detecting and diagnosing disease. This study demonstrates that human gliomas are distinguishable from control tissue on the basis of IR image used in combination with chemometric imaging processing. Using an iterative two-step algorithm – comprised of a linear discriminant analysis guided genetic optimal spectral region selection – tissue types can be discriminated from one another thus providing insight into the malignancy grade of the tissue. A series of classification models were built using a k-fold cross validation scheme and the classification predictions from the various models were combined to provide an aggregated prediction. The validation of the aggregated model reveals an improvement in the classification success rate to 64%.
Mid and far-infrared vibrational spectra of Indometacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid], Diclofenac [[2-(2,6-dichlorophenyl)amino]-benzeneacetic acid] and Niflumic acid [2-3((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid] have been measured at room and low temperatures and analyzed by means of ab initio calculations. The conformational space of these compounds has been scanned using molecular dynamics and complemented with functional density calculations that optimize the geometry of the lowest-energy conformers of each species as obtained in the simulations. The vibrational frequencies were assigned using functional density calculations. The molecular electrostatic potential maps (MEPs) were obtained and analyzed and the corresponding topological study was performed in the frame of the Bader's theory (atoms in molecules).
The interaction of hexa-2,4-dienylideneisopropylamine and 3-phenylprop-2-enylideneisopropylamine with phenol derivatives (pKa = 10.30−0.4) was investigated in solution by Fourier transform IR spectrometry. The influence of hydrogen bond formation on the frequency and intensity of the CN stretching vibration is discussed. The thermodynamic parameters for the normal hydrogen bonds and for the proton transfer complexes were determined; 50% proton transfer is achieved for ΔpKa values of about 2 for both imine systems. The lowest OH stretching frequencies corresponding to a delocalized proton are observed at about the same ΔpKa values.