Characterization of individual atmospheric particles by element mapping in electron probe microanalysis

In this paper procedures for the characterization of individual aerosol particles by element mapping in the electron microprobe are presented. The number, size and qualitative chemical composition of particles is derived from a combination of secondary or backscattered electron images and element distribution maps. Accuracy of the size distribution and reliability of the qualitative analysis procedure were checked with silicate samples. In order to obtain a semi-quantitative estimate of the chemical composition of individual particles the count rates taken from element distribution maps are corrected for matrix and geometric effects using particle ZAF procedures.Dedicated to Professor Dr. rer. nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

Quantitative chemical phase imaging by means of energy filtering transmission electron microscopy

Electron spectroscopic imaging (ESI) in the transmission electron microscope (TEM) is a powerful method to produce 2-dimensional elemental distribution maps. These maps show in a clear way the chemical situation of a small specimen region. In this work we used a Gatan Imaging Filter (GIF) attached to a 200 kV TEM to investigate a Ba-Nd-titanate ceramic. The three phases occuring in this material could be visualized using inner-shell ionization edges (Ba M₄₅, Nd M₄₅ and Ti L₂₃). We applied different image correlation techniques to the ESI elemental maps for direct visualization of the chemical phases. First we simply overlaid the elemental maps assigning each element one colour to form an RGB image. Secondly we used the technique of scatter diagrams to classify the different phases. Finally we quantified the elemental maps by dividing them and multiplying them by the appropriate inner-shell ionization cross-sections which gave atomic ratio images. By using these methods we could clearly identify and quantify the various phases in the Ba-Nd-titanate specimen.Dedicated to Professor Dr. rer. nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

High-speed line-focus Raman microscopy with spectral decomposition of mouse skin

Line-focus Raman microscope system was designed and constructed for high speed quantitative analysis of Raman spectral maps. Laser line generator lens was used for formation of homogeneous laser power distribution thought the line. Three-dimensional Raman maps of mouse skin were obtained using point and line illumination modes. It was shown that laser line illumination can provide Raman spectrum with signal to noise ratio comparable to point illumination mode. The speed of scanning in line-focus mode achieved is two orders of magnitude faster compared to the point illumination mode, which enables ex vivo measurements of large areas of skin surface during a few minutes. Non-negative least squares (NNLS) decomposition of mouse skin spectral maps was done using keratin, lipids, water, lactate, urea and natural moisture factor (NMF) components as library spectra. Fluorescence background of measured spectra was corrected using the fluorescence profile obtained from time-lapse Raman skin measurements, which was used as additional component in NNLS decomposition procedure. The lateral and depth distribution of major skin components obtained from Raman maps mainly correlates with histological information. High-quality line-focus Raman maps from large sample area expand the possibilities of studying skin chemical components distribution.     

Study of the similarity between distribution maps of concentration in near-infrared spectroscopy chemical imaging obtained by different multivariate calibration approaches

Nowadays, near-infrared spectroscopy chemical imaging (NIR-CI) has been widely used in pharmaceutical analysis since it provides important surface information about the samples. In this work the information of NIR-CI at the pixel level was compared through calculation of the similarity between distribution maps of concentration obtained by different multivariate calibration approaches. The comparison was performed by using four different multivariate methods (MCR, MLR, CLS and PLS) in analysis of carbamazepine pharmaceutical formulations. For global determination, all models developed showed RMSEP below 1.9% (w/w) for active principal ingredient (API) and better than 4.6% (w/w) for excipients. Also, the distribution maps obtained by PLS, CLS and MCR showed great similarity for all compounds of the formulation as well with concentrations in the tablets. However, comparing the distribution maps obtained by MLR with those from the other chemometric tools, a lower similarity was observed. Thus, this fitted model does not ensure, by itself, that the images obtained are reliable or accurate. The paper also compares the distribution maps of concentrations obtained from all constituents present in the pharmaceutical formulation with their respective micrographs.     

Application of Piloyan's method in the study of the energetics of a glass-to-crystal transition

On the basis of the results obtained for eutectic Te-Ge glass, the application of the simple Piloyan method for study of the energetics of the glass-to-crystal transition was considered. Some new applications of this method to not yet completed transitions were proposed. Limitations of the method and factors exerting an effect on the calculated activation energies were discussed. Piloyan's procedure was compared with Kissinger's method.The author is very grateful to Dr. Mireille Harmelin for inspiring discussion and suggestions concerning methods for the determination of the activation energy. Financial support of this work by the U. S. National Science Foundation under the Grant GF 42176 is greatly appreciated.     

A method to study the electric field distribution on sample surfaces in atom probe analysis

We report the development of a nondestructive method to estimate the electric field (EF) distribution on a nano-sized sample surface in atom probe (AP) analysis. The simulated EF distribution on an ideal hemisphere indicates that the largest EF exists on the geometrical top of the ideal hemisphere and that EF decreases as the emitting area getting away from the sample apex. To estimate the EF distribution on a real sample surface, the sample apex is determined via comparing the field ion microscopy (FIM) signal intensity of {113} planes on the symmetrical sample surface. A series of contour maps showing the intensity of the evaporated ions (eg, H⁺) was obtained by applying various EFs on the sample surface. A plot of relative EFs with respect to the emitting angle can thus be extracted.     

Resolution and segmentation of hyperspectral biomedical images by multivariate curve resolution-alternating least squares

MCR-ALS is a resolution method that has been applied in many different fields, such as process analysis, environmental data and, recently, hyperspectral image analysis. In this context, the algorithm provides the distribution maps and the pure spectra of the image constituents from the sole information in the raw image measurement. Based on the distribution maps and spectra obtained, additional information can be easily derived, such as identification of constituents when libraries are available or quantitation within the image, expressed as constituent signal contribution. This work summarizes first the protocol followed for the resolution on two examples of kidney calculi, taken as representations of images with major and minor compounds, respectively. Image segmentation allows separating regions of images according to their pixel similarity and is also relevant in the biomedical field to differentiate healthy from non-healthy regions in tissues or to identify sample regions with distinct properties. Information on pixel similarity is enclosed not only in pixel spectra, but also in other smaller pixel representations, such as PCA scores. In this paper, we propose the use of MCR scores (concentration profiles) for segmentation purposes. K-means results obtained from different pixel representations of the data set are compared. The main advantages of the use of MCR scores are the interpretability of the class centroids and the compound-wise selection and preprocessing of the input information in the segmentation scheme.     

Cubane derivatives 1. Synthesis and molecular structures of the esters of 1,4-cubanedicarboxylic acid

An effective method of esterification of 1,4-cubanedicarboxylic acid with alkylsulfuric acids is proposed. The derivatives of alkanols with substantially different pK_a Valus are obtained in high yields. Molecular structures of the esters obtained are confirmed by various methods. The X-ray study showed a remarkable effect of the nature of an alkyl radical on the geometry of the cubane core.Translated fromIzyestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp.668–672, April, 1994.The authors thank Dr. G. V. Lagodzinskaya for analyzing the NMR spectra and for useful discussions.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.     

Decoding two-dimensional polyacrylamide gel electrophoresis complex maps by autocovariance function: a simplified approach useful for proteomics

This paper describes a mathematical approach applied for decoding the complex signal of two-dimensional polyacrylamide gel electrophoresis maps of protein mixtures. The method is helpful in extracting analytical information since separation of all the proteins present in the sample is still far from being achieved and co-migrating proteins are generally present in the same spot. The simplified method described is based on the study of the 2-D autocovariance function (2D-ACVF) computed on an experimental digitized map. The first part of the 2D-ACVF allows for the estimation of the number of proteins present in the sample (2D-ACVF computed at the origin) and of the separation performance (mean spot size). Moreover, the 2D-ACVF plot is a powerful tool in identifying order in the spot position, and singling it out from the complex separation pattern. This method was validated on synthetic maps obtained by computer simulation to describe 2-D PAGE real maps and reference maps retrieved from the SWISS-2DPAGE database. The results obtained are discussed by focusing on specific information relevant in proteomics: sample complexity, separation performance, and identification of spot trains related to post-translational modifications.     

The ‘Theorem of common kinetic runs’ in homogeneous non-isothermal reaction kinetics

The kinetic features of the fundamental homogeneous two-reaction models for linearly increasing temperature have been studied systematically by the analysis of 2500 signal curves (organized in 250 series), obtained from computer simulations and compared with numerous TA experiments.For methods based on a proportionality between the particular reaction rates and their contribution to the derivative overall signal, it has been found that characteristicbehaviour sets of three order-dependent parameters which refer to definite intervals of the starting concentration in series, can be extracted from the halfwidth and shape index of the curves. Such thermoanalytical patterns agree with those obtained from the distribution lists of some thousand DTA- and UV-experiments. They are presented for the 21 most important two-step model and almost independent of the activation data and of the method-dependent proportionality factors. Therefore, they allow for the direct kinetic identification of the complex reaction involved.The continuous interest and the stimulating ideas of Prof. Dr. O. E. Polansky over the twenty years of our collaboration and the subsequent generous support of this work by Prof. Dr. D. Schulte-Frohlinde are gratefully acknowledged. Apart from the assistance of many coworkers, appreciated in the respective articles, I am very obliged to Prof. Dr. R. J. Field for his suggestions for the numerical integrations and having sent a Gear sub-routine; to Prof. Dr. E. Körös for his proposals concerning the analytical techniques; and to W. Angenendt, Dr. B. Weimann and Prof. Dr. E. Ziegler of the joint computer department of both Max-Planck-Institutes in Mülheim for stimulating discussions and help in programming.     

Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis

A method intended to serve as a multivariate quality control tool in the production of pharmaceutical proteins is presented. The method is based on multivariate analysis of peptide maps generated with liquid chromatography-mass spectrometry (LC-MS). Lactate dehydrogenase (LDH) from different species and tissues were used as model compounds in the study. The proteins were digested with Endoproteinase Lys-C before the LC-MS analysis. After data pretreatment of the peptide maps, successful classification of the LDHs were obtained by discriminant analysis with partial least squares regression and artificial neural networks. Further, principal component analysis was applied to visualize the relationships between the samples.     

Temporal ratiometry to assess dynamic concentration distributions of fluorescent molecules in single live cells during continuous diffusional dosing

The introduction of specific molecules into live cells is a widely used approach to probe cellular mechanisms. Recently, we have reported on the sustained dosing of molecules into single cells via a microscopic diffusion port. Here we describe temporal ratiometry, a method to reconstruct intracellular concentration distribution of the delivered molecules as it varies in time during dosing. To characterize this method, we analyzed fluorescence intensity maps obtained during delivery of Lucifer Yellow CH, LY, a polar fluorophore into A7r5 vascular smooth muscle cells, normal rat kidney epithelial cells (NRKE), and MCF-7 human breast cancer cells. Temporal ratiometry indicates a linear increase in concentration of LY in these cells with a nearly uniform distribution during 20 min of delivery. The method cancels the effects of varying cell height and variable accessible volume on the measured intensities at different locations within the cell. Temporal ratiometry will be useful to estimate dynamic changes in intracellular concentration distributions and thus, facilitate the understanding of transport, binding, sequestration, and efflux of molecules introduced into cells.     

Sum frequency generation imaging microscopy of CO on platinum

Sum frequency vibrational spectroscopy is utilized as an imaging technique to distinguish and compare the local response of carbon monoxide (CO) covered platinum (Pt) polycrystalline surface versus the average response of the investigated area. The Pt electrode was prepared using the standard method and was exposed to approximately 1 atm of CO(g). SFG images and vibrational spectra were obtained where the contrast is based on the intrinsic nature of each peak in the CO vibrational spectrum. The illustration of the images and the chemical maps of CO on the platinum surface showed the distribution of the CO across the observed area. The results obtained by comparing the local and the average response confirmed the spatial distributions of the CO on the platinum sample which are due to several reasons such as dipole-dipole coupling and surface coverage. This finding has a significant contribution toward recognizing that surfaces usually considered homogeneous may in fact be quite heterogeneous.     

Density-difference maps in quantum chemistry

Electron density-difference maps, used to study the changes that occur when a molecule changes its state or when the nuclei of a molecule change their relative positions, are generally useful only if the atomic densities cancel when one molecular density distribution is subtracted from the other. When, as in the case of the nonrigid internal rotation in ethane, such a cancellation of atomic densities is not possible the method of simple subtraction is no longer appropriate. It is shown that useful density-difference maps can nevertheless be obtained, when the changes in geometric parameters are small, by the calculation of two generalized density-difference functions: a point difference function which allows a comparison of the densities at corresponding points in the two systems, and a volume difference function to compare the amounts of charge in corresponding regions. The method is illustrated by consideration of a change in bond length of the nitrogen molecule and by the nonrigid internal rotation in ethane.Presented at the XIVth Quantum Theory Conference in Canterbury, England, September 1981.     

Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths

In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.     

New approach based on fuzzy logic and principal component analysis for the classification of two-dimensional maps in health and disease. Application to lymphomas

Two-dimensional (2D) electrophoresis is the most wide spread technique for the separation of proteins in biological systems. This technique produces 2D maps of high complexity, which creates difficulties in the comparison of different samples. The method proposed in this paper for the comparison of different 2D maps can be summarised in four steps: (a) digitalisation of the image; (b) fuzzyfication of the digitalised map in order to consider the variability of the two-dimensional electrophoretic separation; (c) decoding by principal component analysis of the previously obtained fuzzy maps, in order to reduce the system dimensionality; (d) classification analysis (linear discriminant analysis), in order to separate the samples contained in the dataset according to the classes present in said dataset. This method was applied to a dataset constituted by eight samples: four belonging to healthy human lymph-nodes and four deriving from non-Hodgkin lymphomas. The amount of fuzzyfication of the original map is governed by the sigma parameter. The larger the value, the more fuzzy theresulting transformed map. The effect of the fuzzyfication parameter was investigated, the optimal results being obtained for sigma = 1.75 and 2.25. Principal component analysis and linear discriminant analysis allowed the separation of the two classes of samples without any misclassification.     

A new integrated statistical approach to the diagnostic use of two-dimensional maps

Two-dimensional (2-D) electrophoresis is a very useful technique for the analysis of proteins in biological tissues. The complexity of the 2-D maps obtained causes many difficulties in the comparison of different samples. A new method is proposed for comparing different 2-D maps, based on five steps: (i) the digitalisation of the image; (ii) the transformation of the digitalised map in a fuzzy entity, in order to consider the variability of the 2-D electrophoretic separation; (iii) the calculation of a similarity index for each pair of maps; (iv) the analysis by multidimensional scaling of the previously obtained similarity matrix; (v) the analysis by classification or cluster analysis techniques of the resulting map co-ordinates. The method adopted was first tested on some simulated samples in order to evaluate its sensitivity to small changes in the spots position and size. The optimal setting of the method parameters was also investigated. Finally, the method was successfully applied to a series of real samples corresponding to the electrophoretic bidimensional analysis of sera from normal and nicotine-treated rats. Multidimensional scaling allowed the separation of the two classes of samples without any misclassification.     

Intracellular concentration map of magnesium in whole cells by combined use of X-ray fluorescence microscopy and atomic force microscopy

We report a novel experimental approach to derive quantitative concentration map of light elements in whole cells by combining two complementary nano-probe methods: X-ray fluorescence microscopy (XRFM) and atomic force microscopy (AFM). The concentration is derived by normalizing point-by-point the elemental (here Mg) spatial distribution obtained by XRFM, by the thickness measured using AFM. The considerable difference between the elemental distribution and the concentration maps indicates that this procedure is essential to obtain reliable information on the role and function of elements in whole cells.     

Large‐Area Elemental Imaging Reveals Van Eyck's Original Paint Layers on the Ghent Altarpiece (1432), Rescoping Its Conservation Treatment

A combination of large‐scale and micro‐scale elemental imaging, yielding elemental distribution maps obtained by, respectively non‐invasive macroscopic X‐ray fluorescence (MA‐XRF) and by secondary electron microscopy/energy dispersive X‐ray analysis (SEM‐EDX) and synchrotron radiation‐based micro‐XRF (SR μ‐XRF) imaging was employed to reorient and optimize the conservation strategy of van Eyck's renowned Ghent Altarpiece. By exploiting the penetrative properties of X‐rays together with the elemental specificity offered by XRF, it was possible to visualize the original paint layers by van Eyck hidden below the overpainted surface and to simultaneously assess their condition. The distribution of the high‐energy Pb‐L and Hg‐L emission lines revealed the exact location of hidden paint losses, while Fe‐K maps demonstrated how and where these lacunae were filled‐up using an iron‐containing material. The chemical maps nourished the scholarly debate on the overpaint removal with objective, chemical arguments, leading to the decision to remove all skillfully applied overpaints, hitherto interpreted as work by van Eyck. MA‐XRF was also employed for monitoring the removal of the overpaint during the treatment phase. To gather complementary information on the in‐depth layer build‐up, SEM‐EDX and SR μ‐XRF imaging was used on paint cross sections to record micro‐scale elemental maps.