Resonant Mie scattering (RMieS) correction applied to FTIR images of biological tissue samples

Recently a resonant Mie scattering (RMieS) correction approach has been developed and demonstrated to be effective for removing the baseline distortions that compromise the raw data in individual spectra. In this paper RMieS correction is extended to FTIR images of a tissue section from biopsy of the human cervical transformation zone and a coronal tissue section of a Wistar rat brain and compared to the uncorrected images. It is shown that applying RMieS correction to FTIR images a) removes baseline distortions from the image spectra and thus reveals previously hidden information on spatial variation of chemical contents within the tissue and b) can lead to improved automatic tissue feature classification through multivariate cluster analysis.     

Crisp and soft multivariate methods visualize individual cell nuclei in Raman images of liver tissue sections

Raman and infrared spectroscopy have been recognized to be promising tools in clinical diagnostics because they provide molecular contrast without external stains. Here, vertex component analysis (VCA) was applied to Raman and Fourier transform infrared (FTIR) images of liver tissue sections and the results were compared with K-means cluster analysis, fuzzy C-means cluster analysis and principal component analysis. The main components of VCA from three Raman images were assigned to the central vein, periportal vein, cell nuclei, liver parenchyma and bile duct. After resonant Mie scattering correction, VCA of FTIR images identified veins, liver parenchyma, cracks, but no cell nuclei. The advantages of VCA in the context of tissue characterization by vibrational spectroscopic imaging are that the tissue architecture is visualized and the spectral information is reconstructed. Composite images were constructed that revealed a high molecular contrast and that can be interpreted in a similar way like hematoxylin and eosin stained tissue sections.     

Univariate method for background correction in liquid chromatography-Fourier transform infrared spectrometry

An univariate method is proposed for background correction in on-line gradient liquid chromatography-Fourier transform infrared (LC-FTIR) spectrometry using acetonitrile:water as mobile phase components. The method is based on the calculation of the ratio of absorbances (AR) at two characteristic wavenumbers for each spectrum. This parameter is subsequently used to locate the most appropriated eluent spectrum within a reference spectra matrix (RSM) to be subtracted from each spectrum included in the sample chromatogram. To correct minor changes in eluent spectra intensity during the elution of analytes, a correction factor (Kf), defined as the ratio of the absorbance of the sample and the selected eluent spectrum at a defined wavenumber was determined. The performance of the procedure was evaluated by correcting an on-line gradient LC-FTIR injection of a mixture of two pesticides (Atrazine and Diuron). Using the AR of the absorbance at 2248.6 and 2256.3 cm(-1) and a Kf at 2248.6 cm(-1), the correlation factors between FTIR spectra extracted at the peak apex from the LC-FTIR chromatogram and those obtained from pure pesticide standards were 0.975 and 0.94 for Atrazine and Diuron, respectively.     

Propagation, beam geometry, and detection distortions of peak shapes in two-dimensional Fourier transform spectra

Using a solution of Maxwell's equations in the three-dimensional frequency domain, femtosecond two-dimensional Fourier transform (2DFT) spectra that include distortions due to phase matching, absorption, dispersion, and noncollinear excitation and detection of the signal are calculated for Bloch, Kubo, and Brownian oscillator relaxation models. For sample solutions longer than a wavelength, the resonant propagation distortions are larger than resonant local field distortions by a factor of approximately L/lambda, where L is the sample thickness and lambda is the optical wavelength. For the square boxcars geometry, the phase-matching distortion is usually least important, and depends on the dimensionless parameter, L sin(2)(beta)Deltaomega/(nc), where beta is the half angle between beams, n is the refractive index, c is the speed of light, and Deltaomega is the width of the spectrum. Directional filtering distortions depend on the dimensionless parameter, [(Deltaomega)w(0) sin(beta)/c](2), where w(0) is the beam waist at the focus. Qualitatively, the directional filter discriminates against off diagonal amplitude. Resonant absorption and dispersion can distort 2D spectra by 10% (20%) at a peak optical density of 0.1 (0.2). Complicated distortions of the 2DFT peak shape due to absorption and dispersion can be corrected to within 10% (15%) by simple operations that require knowledge only of the linear optical properties of the sample and the distorted two-dimensional spectrum measured at a peak optical density of up to 0.5 (1).     

On recording the true absorption spectrum and the scattering spectrum of a turbid sample: application to cell suspensions of the cyanobacterium Anabaena variabilis

An integrating sphere is often used for recording the absorption spectrum of a turbid sample. If the sample is placed inside the sphere, scattering losses are eliminated, but the recorded spectrum suffers from other distortions. These distortions can be avoided by positioning the sample outside the sphere; but, since some of the scattered light escapes the detector, the recorded spectrum suffers from residual scattering losses. A method proposed by Latimer and Eubanks more than 30 years ago (Arch. Biochem. Biophys. 98 (1962) 274), is put to a quantitative examination, which has shown that one can obtain, by recording two spectra at different distances from the sphere, not only the true absorption spectrum but also the scattering spectra of the sample. Conditions for the validity of the basic assumption underlying the method are investigated by examining suspensions containing various concentrations of cells of the cyanobacterium Anabaena variabilis, and it is shown that the calculated absorbance is proportional to the number density of the cells. The application of the method for quantitative spectrophotometric analysis of pigments in cell suspensions is discussed.     

Line shape distortion effects in infrared spectroscopy

This paper explores different phenomena that cause distortions of infrared absorption spectra by mixing of reflective and absorptive band shape components of infrared spectra, and the resulting distortion of observed band shapes. In the context of this paper, we refer to the line shape of the variations of the refractive index in spectral regions of an absorption maximum (i.e., in regions of "anomalous dispersion") as "dispersive" or "reflective" line shape contributions, in analogy to previous spectroscopic literature. These distortions usually result in asymmetric bands with a negative intensity contribution at the high wavenumber of the band, accompanied by a shift toward lower wavenumber, and confounded band intensities. In extreme cases of band distortions caused by the "resonance Mie" (RMie) mechanism, spectral peaks may be split into doublets of peaks, change from positive to negative peaks, or appear as derivative-shaped features.     

Modifying infrared scattering effects of single yeast cells with plasmonic metal mesh

The scattering effects in the infrared (IR) spectra of single, isolated bread yeast cells (Saccharomyces cerevisiae) on a ZnSe substrate and in metal microchannels have been probed by Fourier transform infrared imaging microspectroscopy. Absolute extinction [(3.4±0.6)×10(-7) cm(2) at 3178 cm(-1)], scattering, and absorption cross sections for a single yeast cell and a vibrational absorption spectrum have been determined by comparing it to the scattering properties of single, isolated, latex microspheres (polystyrene, 5.0 μm in diameter) on ZnSe, which are well modeled by the Mie scattering theory. Single yeast cells were then placed into the holes of the IR plasmonic mesh, i.e., metal films with arrays of subwavelength holes, yielding "scatter-free" IR absorption spectra, which have undistorted vibrational lineshapes and a rising generic IR absorption baseline. Absolute extinction, scattering, and absorption spectral profiles were determined for a single, ellipsoidal yeast cell to characterize the interplay of these effects.     

Optimization of the Thermal Radiation Extinction of Silicon Carbide in a Silica Powder Matrix

In order to optimize the infrared extinction of a SiC-powder in a silica powder matrix, Mie scattering calculations for spherical SiC-particles have been performed. A single oscillator-model was applied to calculate the optical constants of SiC. Taking into account the particle size distribution of a commercially available SiC-powder, its wavelength dependent extinction coefficient was calculated. The result is in very good agreement with the extinction spectrum of the powder derived by infrared optically measurements. Mie scattering theory also was used to find the optimum mean SiC-particle diameter of a mixture of 20% SiC-powder and 80% silica powder. This revised version was published online in July 2006 with corrections to the Cover Date.     

Raman Scattering at Resonant or Near-Resonant Conditions: A Generalized Short-Time Approximation

We investigate the dynamics of resonant Raman scattering in the course of the frequency de-tuning. The dephasing in the time domain makes the scattering fast when the photon energy is tuned from the absorption resonance. This makes frequency detuning to act as a cam-era shutter with a regulated scattering duration and provides a practical tool of controlling the scattering time in ordinary stationary measurements. The theory is applied to resonant Raman spectra of a couple of few-mode model systems and to trans-1,3,5-hexatriene and guanine-cytosine (G-C) Watson-Crick base pairs (DNA) molecules. Besides some particular physical effects, the regime of fast scattering leads to a simplification of the spectrum as well as to the scattering theory itself. Strong overtones appear in the Raman spectra when the photon frequency is tuned in the resonant region, while in the mode of fast scattering, the overtones are gradually quenched when the photon frequency is tuned more than one vibra-tional quantum below the first absorption resonance. The detuning from the resonant region thus leads to a strong purification of the Raman spectrum from the contamination by higher overtones and soft modes and purifies the spectrum also in terms of avoidance of dissociationand interfering fluorescence decay of the resonant state. This makes frequency detuning a very useful practical tool in the analysis of the resonant Raman spectra of complex systems and considerably improves the prospects for using the Raman effect for detection of foreign substances at ultra-low concentrations.     

FT-IR, FT-Raman spectra and ab initio DFT vibrational analysis of p-bromophenoxyacetic acid

The Fourier transform Raman and Fourier transform infrared spectra of p-bromophenoxyacetic acid were recorded in the solid phase. The equilibrium geometry, harmonic vibrational frequencies, infrared intensities and Raman scattering activities were calculated by HF and DFT (B3LYP) method with the 6-31G(d,p) basis set. The scaled theoretical wavenumbers showed very good agreement with the experimental ones. A detailed interpretation of the infrared and Raman spectra of p-bromophenoxyacetic acid is reported on the basis of the calculated potential energy distribution. The theoretical spectrograms for the IR spectrum of the title molecule have been constructed.     

Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll

A pulse sequence of z-restored spin echo, -pi-beta-tau-pi-tau-, employing a pi pulse in the middle of the delay (2tau) to form a spin echo and the two pi pulses together to restore the residual longitudinal magnetization back to + z direction, is described. (13)C spectra of organic compounds provide a wealth of structural information; however, (13)C 1D spectra acquired using reverse geometry probes can have significant baseline humps or rolls because of pulse ring-down within the coil. The baseline distortions are especially apparent in spectra acquired using cryogenically enhanced probes. The baseline problem may be alleviated by extending the delay between the last pulse and the starting point of acquisition. However, uses of long delay times introduce large negative first-order phase corrections which themselves produce baseline roll. The prescribed experiment can be used to completely remove the hump, roll or dip in the baseline of the (13)C spectrum and at the same time obtain sensitivity similar to the experiment of a single beta pulse. We believe that this experiment will be of general applications in acquiring high-quality (13)C NMR data with reverse geometry probes and spectral interpretation.     

Fourier-transform mid-infrared FPA imaging of a complex multicellular nematode

Here we report on the application of mid-IR imaging to a complex multicellular organism, a nematode. Despite the increase in sample complexity and sample thickness from previously measured cells and tissue sections, it is still possible, through the application of hierarchical cluster analysis, to elucidate biochemical information and spatial distribution information that can be linked to different tissue types in the nematode, namely somatic and non-striated muscle and the collagen rich cuticle. Comparison of HCA clusters from individuals from different species shows clusters can be associated specifically to a particular species, but that few clusters are shared between individuals from different species. In addition, using a synthetic fibre as a model for the nematode, we also show that the IR spectra can be affected by Mie scattering type baseline oscillations which are especially severe towards the edges of the fibre/nematode, but that these can be, to some extent, corrected for using currently available correction algorithms prior to cluster analysis.     

Computer-assisted multicomponent spectral analysis with fuzzy data sets

A new approach to the interpretation of spectra with “fuzzy sets” is described. A computer program CIF (Compound Identification with Fuzzy sets) is applied. This program is capable of finding components in a mixture by comparing the sample spectrum with reference spectra in a library. The applications discussed involve the interpretation of infrared spectra. The problems of spectral library search are discussed, an elementary introduction to fuzzy set theory is given, and applications to spectral library search are demonstrated.     

Mass Spectral Peak Distortion Due to Fourier Transform Signal Processing

Distortions of peaks can occur when one uses the standard method of signal processing of data from the Orbitrap and other FT-based methods of mass spectrometry. These distortions arise because the standard method of signal processing is not a linear process. If one adds two or more functions, such as time-dependent signals from a Fourier transform mass spectrometer and performs a linear operation on the sum, the result is the same as if the operation was performed on separate functions and the results added. If this relationship is not valid, the operation is non-linear and can produce unexpected and/or distorted results. Although the Fourier transform itself is a linear operator, the standard algorithm for processing spectra in Fourier transform-based methods include non-linear mathematical operators such that spectra processed by the standard algorithm may become distorted. The most serious consequence is that apparent abundances of the peaks in the spectrum may be incorrect. In light of these considerations, we performed theoretical modeling studies to illustrate several distortion effects that can be observed, including abundance distortions. In addition, we discuss experimental systems where these effects may manifest, including suggested systems for study that should demonstrate these peak distortions. Finally, we point to several examples in the literature where peak distortions may be rationalized by the phenomena presented here. Graphical Abstract

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Influence and correction of peak shift and band broadening observed by rank analysis on vibrational bands from variable-temperature measurements

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.     

Vibrational spectra and assignments of 2-amino-5-iodopyridine by ab initio Hartree-Fock and density functional methods

The Fourier transform Raman and Fourier transform infrared spectra of 2-amino-5-iodopyridine were recorded in the solid phase. The equilibrium geometry, harmonic vibrational frequencies, infrared intensities and Raman scattering activities were calculated by HF and DFT (B3LYP) methods with the 6-31G(d,p) basis set for C, N, H and LANL2DZ pseudopotential for I. The scaled theoretical wavenumbers showed very good agreement with the experimental ones. A detailed interpretation of the infrared and Raman spectra of 2-amino-5-iodopyridine is reported on the basis of the calculated potential energy distribution. The theoretical spectrograms for the IR spectrum of the title molecule have been constructed.     

Distinction between some saccharides in scattered optical sum frequency intensity images

Using an optical sum frequency (SF) microscope with visible and infrared light pulses for excitation, we have attempted to distinguish among four saccharide species. The saccharides we studied were d-glucose, amylopectin, beta-cyclodextrin, and amylose. The wavelength of the infrared light was resonant to CH vibration. Amylose showed very weak sum frequency scattering for CH vibration among the four saccharides. As for the other three saccharides, we found a big difference in the sum frequency spectra of their CH stretching vibration near 2900 cm-1, when the incident visible and infrared light pulses were p- and s-polarized, respectively. Based on these facts, we have demonstrated a distinction between these three saccharides in the scattered SF intensity images.     

Completely automated open-path FT-IR spectrometry

Atmospheric analysis by open-path Fourier-transform infrared (OP/FT-IR) spectrometry has been possible for over two decades but has not been widely used because of the limitations of the software of commercial instruments. In this paper, we describe the current state-of-the-art of the hardware and software that constitutes a contemporary OP/FT-IR spectrometer. We then describe advances that have been made in our laboratory that have enabled many of the limitations of this type of instrument to be overcome. These include not having to acquire a single-beam background spectrum that compensates for absorption features in the spectra of atmospheric water vapor and carbon dioxide. Instead, an easily measured “short path-length” background spectrum is used for calculation of each absorbance spectrum that is measured over a long path-length. To accomplish this goal, the algorithm used to calculate the concentrations of trace atmospheric molecules was changed from classical least-squares regression (CLS) to partial least-squares regression (PLS). For calibration, OP/FT-IR spectra are measured in pristine air over a wide variety of path-lengths, temperatures, and humidities, ratioed against a short-path background, and converted to absorbance; the reference spectrum of each analyte is then multiplied by randomly selected coefficients and added to these background spectra. Automatic baseline correction for small molecules with resolved rotational fine structure, such as ammonia and methane, is effected using wavelet transforms. A novel method of correcting for the effect of the nonlinear response of mercury cadmium telluride detectors is also incorporated. Finally, target factor analysis may be used to detect the onset of a given pollutant when its concentration exceeds a certain threshold. In this way, the concentration of atmospheric species has been obtained from OP/FT-IR spectra measured at intervals of 1 min over a period of many hours with no operator intervention.     

Infrared spectrum of nitric acid dihydrate: Influence of particle shape

In situ Fourier transform infrared (FTIR) extinction spectra of airborne alpha-NAD microparticles generated by two different methods were recorded in the large coolable aerosol chamber AIDA of Forschungszentrum Karlsruhe. The extinction spectrum of alpha-NAD crystals obtained by shock freezing of a HNO3/H2O gas mixture could be accurately reproduced using Mie theory with published refractive indices of alpha-NAD as input. In contrast, Mie theory proved to be inadequate to properly reproduce the infrared extinction spectrum of alpha-NAD crystals which were formed via homogeneous nucleation of supercooled HNO3/H2O solution droplets, evaporating slowly on a time scale of several hours at about 195 K. Much better agreement between measured and calculated extinction spectra was obtained by T-matrix calculations assuming oblate particles with aspect ratios greater than five. This indicates that strongly aspherical alpha-NAD crystals are obtained when supercooled nitric acid solution droplets freeze and grow slowly, a process which has been discussed as a potential pathway to the formation of crystalline polar stratospheric cloud (PSC) particles.     

Time dependence of the resonant and near resonant photon moleculaar interactions

Using scattering theory we have derived an explicit expression for the time dependence of the resonant interaction of lorentzian photons with molecular systems. A discussion is presented on the dependence of molecular states on the nature of the exciting photon with an additional regard for the appropriate choice of basis sets. By examining the time dependence of the scattered photon a clear resonance Raman scattering resonance fluorecence limit emerges based on the temporal relationship of the scattered photon to that of the resonant state and incident photon. The influence on the resonant scattering of random fluctuation and relaxation processes that lead to dissipation is discussed with reference to scattering experiments performed in the gas and condensed phases.