Interactive hyperspectral approach for exploring and interpreting DESI-MS images of cancerous and normal tissue sections

Desorption electrospray ionization (DESI) is an ambient mass spectrometry (MS) technique that can be operated in an imaging mode. It is known to provide valuable information on disease state and grade based on lipid profiles in tissue sections. Comprehensive exploration of the spatial and chemical information contained in 2D MS images requires further development of methods for data treatment and interpretation in conjunction with multivariate analysis. In this study, we employ an interactive approach based on principal component analysis (PCA) to interpret the chemical and spatial information obtained from MS imaging of human bladder, kidney, germ cell and prostate cancer and adjacent normal tissues. This multivariate strategy facilitated distinction between tumor and normal tissue by correlating the lipid information with pathological evaluation of the same samples. Some common lipid ions, such as those of m/z 885.5 and m/z 788.5, nominally PI(18 : 0/20 : 4) and PS(18 : 0/18 : 1), as well as ions of free fatty acids and their dimers, appeared to be highly characterizing for different types of human cancers, while other ions, such as those of m/z 465.5 (cholesterol sulfate) for prostate cancer tissue and m/z 795.5 (seminolipid 16 : 0/16 : 0) for germ tissue, appeared to be extremely selective for the type of tissue analyzed. These data confirm that lipid profiles can reflect not only the disease/health state of tissue but also are characteristic of tissue type. The manual interactive strategy presented here is particularly useful to visualize the information contained in hyperspectral MS images by automatically connecting regions of PCA score space to pixels of the 2D physical object. The procedures developed in this study consider all the spectral variables and their inter-correlations, and guide subsequent investigations of the mass spectra and single ion images to allow one to maximize characterization between different regions of any DESI-MS image.     

Visualisation and characterisation of ageing induced changes of polymeric surfaces by spectroscopic imaging methods

A polymeric resin material was chosen as the model system to visualise the ageing-induced chemical surface changes with molecular spectroscopic imaging techniques and correlate these results to physical properties such as colour changes. The influence of light radiation, temperature and humidity on the polymeric surfaces was analysed by means of attenuated total reflection infrared imaging, Raman imaging spectroscopy and scanning electron microscopy. Samples were analysed before, during and after the weathering/ageing tests. From these combined data, the mechanisms for the damaging of the resin surface under the various environmental conditions (as applied in the accelerated ageing tests) were deduced. Photo-oxidative decay of the resin leading to a degradation of the uppermost surface layers as well as hydrolysis of the aged surface was identified. The combination of the spectral and spatial data as obtained from spectroscopic imaging with the morphological and elemental information of scanning electron microscopic mapping experiments turned out to be highly advantageous for the elucidation of ageing processes. A correlation between the molecular spectroscopic data and the results from the macroscopic colour difference measurements was found.     

Super-resolution and Raman chemical imaging: from multiple low resolution images to a high resolution image

Imaging in Raman spectroscopy is a valuable tool for analytical chemistry. Although molecular characterization at micron level is achieved for many applications, it usually fails producing chemical images of micron size samples as expected in chemical, environmental and biological analysis. The aim of the work is to introduce the potential of super-resolution in vibrational spectroscopic imaging. This original chemometrics approach uses several low resolution images of the same sample in order to retrieve a higher resolution chemical image. It is thus possible to overcome in a certain way some physical and instrumentals limitations. To illustrate the methodology, sub-micronic details of a Si/Au sample are retrieved from low resolution images with different super-resolution algorithms. The better results are obtained with Iterative L2/Bilateral Total Variation regularization method. The use of a regularization procedure gives also better results since its first property is to preserve edges during the reconstruction of the super-resolved image. This concept of chemical image data processing should open new analytical opportunities.     

3D FT-IR imaging spectroscopy of phase-separation in a poly(3-hydroxybutyrate)/poly(l-lactic acid) blend

In the present study, 3D FT-IR spectroscopic imaging measurements were applied to study the phase separation of a poly(3-hydroxybutyrate) (PHB)/poly(l-lactic acid) (PLA) (50:50 wt.%) polymer blend film. While in 2D projection imaging the z-dependent information is overlapped, thereby complicating the analysis, FT-IR spectro-micro-tomography, obtained from computed tomographic back projection calculations, results in distinct 3D chemical images that provide detailed information of phase separation of the two polymer components that are well separated.     

Classification of malignant gliomas by infrared spectroscopic imaging and linear discriminant analysis

Infrared (IR) spectroscopy provides a sensitive molecular fingerprint for tissue without external markers. Supervised classification models can be trained to identify the tissue type based on the spectroscopic fingerprint. Infrared imaging spectrometers equipped with multi-channel detectors combine the spectral and spatial information. Tissue areas of 4 x 4 mm(2) can be analyzed within a few minutes in the macroscopic imaging mode. An approach is described to apply this methodology to human astrocytic gliomas, which are graded according to their malignancy from one to four. Multiple IR images of three tissue sections from one patient with a malignant glioma are acquired and assigned to the six classes normal brain tissue, astrocytoma grade II, astrocytoma grade III, glioblastoma multiforme grade IV, hemorrhage, and other tissue by a linear discriminant analysis model which was trained by data from a single-channel detector. Before the model is applied here, the spectra are shown to be virtually identical. The first specimen contained approximately 95% malignant glioma regions, that means astrocytoma grade III or glioblastoma. The smaller percentage of 12-34% malignant glioma in the second specimen is consistent with its location at the tumor periphery. The detection of less than 0.2% malignant glioma in the third specimen points to a location outside the tumor. The results were correlated with the cellularity of the tissue which was obtained from the histopathologic gold standard. Potential applications of IR spectroscopic imaging as a rapid tool to complement established diagnostic methods are discussed.     

Infrared chemical imaging: Spatial resolution evaluation and super-resolution concept

Chemical imaging systems help to solve many challenges in various scientific fields. Able to deliver rapid spatial and chemical information, modern infrared spectrometers using Focal Plane Array detectors (FPA) are of great interest. Considering conventional infrared spectrometers with a single element detector, we can consider that the diffraction-limited spatial resolution is more or less equal to the wavelength of the light (i.e. 2.5-25 μm). Unfortunately, the spatial resolution of FPA spectroscopic setup is even lower due to the detector pixel size. This becomes a real constraint when micron-sized samples are analysed. New chemometrics methods are thus of great interest to overcome such resolution drawback, while keeping our far-field infrared imaging spectrometers. The aim of the present work is to evaluate the super-resolution concept in order to increase the spatial resolution of infrared imaging spectrometers using FPA detectors. The main idea of super-resolution is the fusion of several low-resolution images of the same sample to obtain a higher-resolution image. Applying the super-resolution concept on a relatively low number of FPA acquisitions, it was possible to observe a 30% decrease in spatial resolution.     

Fourier transform infrared imaging analysis in discrimination studies of St. John's wort (Hypericum perforatum)

In the present study, Fourier transform infrared (FTIR) imaging and data analysis methods were combined to study morphological and molecular patterns of St. John's wort (Hypericum perforatum) in detail. For interpretation, FTIR imaging results were correlated with histological information gained from light microscopy (LM). Additionally, we tested several evaluation processes and optimized the methodology for use of complex FTIR microscopic images to monitor molecular patterns. It is demonstrated that the combination of the used spectroscopic method with LM enables a more distinct picture, concerning morphology and distribution of active ingredients, to be gained. We were able to obtain high-quality FTIR microscopic imaging results and to distinguish different tissue types with their chemical ingredients.     

High-resolution transmission electron microscopy: the ultimate nanoanalytical technique

To be able to determine the elemental composition and morphology of individual nanoparticles consisting of no more than a dozen or so atoms that weigh a few zeptograms (10(-21) g) is but one of the attainments of modern electron microscopy. With slightly larger specimens (embracing a few unit cells of the structure) their symmetry, crystallographic phase, unit-cell dimension, chemical composition and often the valence state (from parallel electron spectroscopic measurements) of the constituent atoms may also be determined using a scanning beam of electrons of ca. 0.5 nm diameter. Nowadays electron crystallography, which treats the digital data of electron diffraction (ED) and high-resolution transmission electron microscope (HRTEM) images of minute (ca. 10(-18)g) specimens in a quantitatively rigorous manner, solves hitherto unknown structures just as X-ray diffraction does with bulk single crystals. In addition, electron tomography (see cover photograph and its animation) enables a three-dimensional picture of the internal structure of minute objects, such as nanocatalysts in a single pore, as well as structural faults such as micro-fissures, to be constructed with a resolution of 1 nm from an angular series of two-dimensional (projected) images. Very recently (since this article was first written) a new meaning has been given to electron crystallography as a result of the spatio-temporal resolution of surface phenomena achieved on a femtosecond timescale.     

Characterization of porcine skin as a model for human skin studies using infrared spectroscopic imaging

Porcine skin is often considered a substitute for human skin based on morphological and functional data, for example, for transdermal drug diffusion studies. A chemical, structural and temporal characterization of porcine skin in comparison to human skin is not available but will likely improve our understanding of this porcine skin model. Here, we employ Fourier transform infrared (FT-IR) spectroscopic imaging to holistically measure chemical species as well as spatial structure as a function of time to characterize porcine skin as a model for human skin. Porcine skin was found to resemble human skin spectroscopically and differences are elucidated. Cryo-prepared fresh porcine skin samples for spectroscopic imaging were found to be stable over time and small variations are observed. Hence, we extended characterization to the use of this model for dynamic processes. In particular, the capacity and stability of this model in transdermal diffusion is examined. The results indicate that porcine skin is likely to be an attractive tool for studying diffusion dynamics of materials in human skin.     

Intra-operative optical diagnostics with vibrational spectroscopy

Established methods for characterization of tissue and diagnostics, for example histochemistry, magnetic resonance imaging (MRI), X-ray tomography, or positron emission tomography (PET), are mostly not suitable for intra-operative use. However, there is a clear need for an intra-operative diagnostics especially to identify the borderline between normal and tumor tissue. Currently, vibrational spectroscopy techniques (both Raman and infrared) complement the standard methods for tissue diagnostics. Vibrational spectroscopy has the potential for intra-operative use, because it can provide a biochemically based profile of tissue in real time and without requiring additional contrast agents, which may perturb the tissue under investigation. In addition, no electric potential needs to be applied, and the measurements are not affected by electromagnetic fields. Currently, promising approaches include Raman fiber techniques and nonlinear Raman spectroscopy. Infrared spectroscopy is also being used to examine freshly resected tissue ex vivo in the operating theater. The immense volume of information contained in Raman and infrared spectra requires multivariate analysis to extract relevant information to distinguish different types of tissue. The promise and limitations of vibrational spectroscopy methods as intra-operative tools are surveyed in this review.     

Synthesis of 11C, 18F, 15O, and 13N radiolabels for positron emission tomography

Positron emission tomography (PET) is a powerful and rapidly developing area of molecular imaging that is used to study and visualize human physiology by the detection of positron-emitting radiopharmaceuticals. Information about metabolism, receptor/enzyme function, and biochemical mechanisms in living tissue can be obtained directly from PET experiments. Unlike magnetic resonance imaging (MRI) or computerized tomography (CT), which mainly provide detailed anatomical images, PET can measure chemical changes that occur before macroscopic anatomical signs of a disease are observed. PET is emerging as a revolutionary method for measuring body function and tailoring disease treatment in living subjects. The development of synthetic strategies for the synthesis of new positron-emitting molecules is, however, not trivial. This Review highlights key aspects of the synthesis of PET radiotracers with the short-lived positron-emitting radionuclides (11)C, (18)F, (15)O, and (13)N, with emphasis on the most recent strategies.     

Chemometrics applied to unravel multicomponent processes and mixtures: Revisiting latest trends in multivariate resolution

Progress in the analysis of multicomponent processes and mixtures relies on the combination of sophisticated instrumental techniques and suitable data analysis tools focused on the interpretation of the multivariate responses obtained. Despite the differences in compositional variation, complexity and origin, the raw measurements recorded in a multicomponent chemical system can be very often described with a simple model consisting of the composition-weighted sum of the signals of their pure compounds.

Multivariate resolution methods have been the tools designed to unravel this pure compound information from the non-selective mixed original experimental output. The evolution of these chemometric approaches through the improvement of exploratory tools, the adaptation to work with complex data structures, the ability to introduce chemical and mathematical information in the algorithms and the better quality assessment of the results obtained is revisited. The active research on these chemometric area has allowed the successful application of these methodologies to chemical problems as complex and diverse as the interpretation of protein folding processes or the resolution of spectroscopic images.     

Nanoscale Chemical Imaging Using Tip‐Enhanced Raman Spectroscopy: A Critical Review

Methods for chemical analysis at the nanometer scale are crucial for understanding and characterizing nanostructures of modern materials and biological systems. Tip‐enhanced Raman spectroscopy (TERS) combines the chemical information provided by Raman spectroscopy with the signal enhancement known from surface‐enhanced Raman scattering (SERS) and the high spatial resolution of atomic force microscopy (AFM) or scanning tunneling microscopy (STM). A metallic or metallized tip is illuminated by a focused laser beam and the resulting strongly enhanced electromagnetic field at the tip apex acts as a highly confined light source for Raman spectroscopic measurements. This Review focuses on the prerequisites for the efficient coupling of light to the tip as well as the shortcomings and pitfalls that have to be considered for TERS imaging, a fascinating but still challenging way to look at the nanoworld. Finally, examples from recent publications have been selected to demonstrate the potential of this technique for chemical imaging with a spatial resolution of approximately 10 nm and sensitivity down to the single‐molecule level for applications ranging from materials sciences to life sciences.     

FT-IR spectroscopic imaging microscopy of wheat kernels using a Mercury–Cadmium–Telluride focal-plane array detector

A 64×64 Mercury–Cadmium–Telluride (MCT) focal-plane array detector attached to a Fourier transform infrared (FT-IR) microscope was used to spectroscopically image 8-μm-thick cross-sections of wheat kernels in the fingerprint region of the infrared spectrum. After fast-Fourier transformation of the raw image interferograms, the data can be displayed as either a series of spectroscopic images collected at individual wavelengths, or as a collection of IR spectra obtained at each pixel position in the image. Image contrast is achieved due to the intrinsic chemical nature of the sample at each pixel location in the image. Individual cell layers near the outer portion of the wheat kernel, as well as the primary root within the germ, can be clearly differentiated in the IR images as a result of this enhanced chemical contrast.     

On-line monitoring of pH junctions in capillary electrophoresis using Fourier transform infrared spectrometry

Fourier transform mid-infrared spectroscopic detection is proposed as an on-line detection technique for the study of on-line preconcentration processes in capillary electrophoresis (CE). The molecule-specific information contained in mid-IR spectra can be used to directly determine the chemical compositions of individual zones and their boundaries. This paper reports on pH junctions employed in myoglobin analysis. On-line mid-IR detection allowed the shape of the sample peak to be monitored as well as the chemical compositions of the surrounding zones. From this information it was possible to obtain detailed insights into the actual chemical compositions of the individual zones governing the efficiency of the preconcentration technique applied. The principle of measurement outlined here can therefore also be regarded as a promising one for investigating other on-line preconcentration techniques, like stacking, sweeping, and pH junction-sweeping among others. Fourier transform mid-infrared spectroscopic detection has been employed in pH junction experiments. This approach can be used to measure the chemical compositions of the phase boundaries formed, as well as the relative positions of the analyte in the zones. The principle of this technique is demonstrated by measuring myoglobin (acetate buffer, pH 4.5) in an ammonium BGE (pH 9.25)     

Combined Fourier-transform infrared imaging and desorption electrospray-ionization linear ion-trap mass spectrometry for analysis of counterfeit antimalarial tablets

This paper reports use of a combination of Fourier-transform infrared (FTIR) spectroscopic imaging and desorption electrospray ionization linear ion-trap mass spectrometry (DESI MS) for characterization of counterfeit pharmaceutical tablets. The counterfeit artesunate antimalarial tablets were analyzed by both techniques. The results obtained revealed the ability of FTIR imaging in non-destructive micro-attenuated total reflection (ATR) mode to detect the distribution of all components in the tablet, the identities of which were confirmed by DESI MS. Chemical images of the tablets were obtained with high spatial resolution. The FTIR spectroscopic imaging method affords inherent chemical specificity with rapid acquisition of data. DESI MS enables high-sensitivity detection of trace organic compounds. Combination of these two orthogonal surface-characterization methods has great potential for detection and analysis of counterfeit tablets in the open air and without sample preparation.     

Use of local rank‐based spatial information for resolution of spectroscopic images

Spectroscopic images are singular chemical measurements that enclose chemical and spatial information about samples. Resolution of spectroscopic images is focused on the recovery of the pure spectra and distribution maps of the image constituents from the sole raw spectroscopic measurement. In image resolution, constraints are generally limited to non‐negativity and the spatial information is generally not used. Local rank analysis methods have been adapted to describe the local spatial complexity of an image, providing specific pixel information. This local rank information combined with reference spectral information allows the identification of absent compounds in pixels with low compound overlap. The introduction of this information in the resolution process under the form of constraints helps to increase the performance of the resolution method and to decrease the ambiguity linked to the final solutions. Copyright © 2008 John Wiley & Sons, Ltd.     

Conformational studies on 1,2-di- and 1,2,3-trisubstituted heterocycles. A spectroscopic and theoretical study of 3-acylaminopicolinic Acid derivatives and their N-oxides.

Theoretical studies involving minimization of model 3-propanoylaminopicolinic acids (10d-trans, 10d-cis), methyl ester (10a), and corresponding -N-oxide derivatives (10b, 10c-trans, 10c-cis) using AM1 gave conformations contrary to both sound chemical intuition and experimental data. RHF ab initio calculations using the 6-31G and 6-31G basis sets on the other hand corroborated spectroscopic data. 3-Amidopicolinic acid derivatives (7a-9a, 7b-9b, 7c-9c, 9d) were prepared and studied by NMR and IR spectroscopy. The results show that a strong intramolecular hydrogen bond between amide-H and the 2-carboxyl substituent results in a planar molecular conformation. This is particularly profound in the 3-acylaminopicolinic acid N-oxides (c-series). When the 2-substituent is a methyl ester on the other hand, repulsion between N-oxide and ester functions induces twisting of the carbomethoxy group out of the plane of the aromatic ring. The type of method used in molecular modeling can have profound impact on the final theoretical result in the case of the above-mentioned class of compounds. Our results indicate, that it is advisable to employ ab initio methods for modeling these types of compounds, and further, that the choice of basis set used for such calculations should depend on the type of information required. Thus, for most purposes pertaining to molecular conformation the 6-31G basis set provides sufficiently sound data in relatively short CPU time. For data related to electronic properties such as involvement of the N-oxide function or spectroscopic information such as IR frequencies or (1)H or (15)N NMR chemical shifts, the use of polarization functions as contained in the 6-31G basis set seems to be a must.