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Colorectal cancers are the third most common type of cancers globally, affecting both sexes [1]. As of now, histopathology is the gold standard method for colon cancer diagnosis. Newer technologies are the important need of the hour to complement the existing approaches, for better understanding the onset and progression of the disease. Infrared (IR) imaging could be a potential candidate method because of its capability to probe non-destructively and in a label-free manner the intrinsic chemical bonds present in the tissue, thus giving a “spectral fingerprint” of its composition and structures. To this end, we have developed IR spectral histopathology with the aims to: (a) examine, the molecular changes between normal and tumoral colon tissues, (b) exploit its potentials to identify new diagnostic markers to complement conventional histopathology, and (c) develop an algorithm as an automatic diagnostic tool for tumor prediction, directly on paraffinized samples, without chemical de-waxing, staining or any further preparation.

Skin hydration plays an important role in the optimal physical properties and physiological functions of the skin. Despite the advancements in the last decade, dry skin remains the most common characteristic of human skin disorders. Thus, it is important to understand the effect of hydration on Stratum Corneum (SC) components. In this respect, our interest consists in correlating the variations of unbound and bound water content in the SC with structural and organizational changes in lipids and proteins using a non-invasive technique: Raman spectroscopy. Raman spectra were acquired on human SC at different relative humidity (RH) levels (4-75%). The content of different types of water, bound and free, was measured using the second derivative and curve fitting of the Raman bands in the range of 3100-3700 cm(-1). Changes in lipidic order were evaluated using νC-C and νC-H. To analyze the effect of RH on the protein structure, we examined in the Amide I region, the Fermi doublet of tyrosine, and the νasymCH3 vibration. The contributions of totally bound water were found not to vary with humidity, while partially bound water varied with three different rates. Unbound water increased greatly when all sites for bound water were saturated. Lipid organization as well as protein deployment was found to be optimal at intermediate RH values (around 60%), which correspond to the maximum of SC water binding capacity. This analysis highlights the relationship between bound water, the SC barrier state and the protein structure and elucidates the optimal conditions. Moreover, our results showed that increased content of unbound water in the SC induces disorder in the structures of lipids and proteins.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer death worldwide. The development of novel diagnostic methods is needed to detect tumours at an early stage when patients are eligible for curative treatments. The purpose of this proof-of-concept study was to determine if micro-Raman spectroscopy applied to the sera of cirrhotic patients may be an alternative method for rapidly discriminating patients with and without HCC. Serum samples were collected from 2 groups of patients: cirrhotic patients with HCC (n = 37) and without HCC (n = 34). Two different approaches were used, dried serum drops and freeze-dried serum, and micro-Raman spectra were acquired in the point-mode with a 785 nm laser excitation in the spectral range of 600-1800 cm(-1). Spectra were quality-tested and pre-processed (smoothing, baseline subtraction, vector normalization). Using principal component analysis, the 2 classes, corresponding to cirrhotic patients with and without HCC, could not be differentiated. In contrast, the support vector machine method using the leave-one-out cross validation procedure was able to correctly classify the two groups of patients with an overall rate of accuracy of 84.5% to 90.2% for dried serum drops and 86% to 91.5% for freeze-dried serum. These results are promising and support the concept that serum micro-Raman spectroscopy may become a useful diagnostic tool to detect biomarkers in the field of cancer, as described here for distinguishing between cirrhotic patients with and without HCC.

Caffeine is utilised as a reference for permeation studies in dermatology and cosmetology. The present work aimed to monitor the permeation of a caffeine solution through the skin. For this purpose, Raman and infrared studies were performed. Raman microspectroscopy permitted a dynamic follow-up of the caffeine diffusion. In complementary, infrared microimaging provided information of the caffeine localization in the skin by applying multivariate statistical processing on skin tissue sections. Herein, we prove the possibility of tracking low concentrations of caffeine through the skin and we highlight some experimental limitations of vibrational spectroscopies.

Dynamic follow-up of exogenous molecules permeation through the skin is one among many competing applications for confocal Raman microspectroscopy. Previous studies showed the feasibility of tracking actives through the skin; the next step should be recording in vivo kinetics. Thus, we conducted a study to evaluate the possibility of detecting low concentrations of caffeine and resveratrol solutions through the skin using confocal Raman microspectroscopy. After topical application of each active on the skin surface, Raman profiles were recorded over nine hours. The challenge was to pursuit these actives respecting the concentration used in some dermatological formulations. Molecules were successfully detected and kinetic profiles were registered over time. The heterogeneity of skin structure and the complexity of molecules diffusion were reflected through the kinetic results.

Cytotoxicity of several classes of antitumor DNA intercalators is thought to result from disturbance of DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here, molecular interactions of the potent antitumor drug amsacrine ( m -AMSA), an inhibitor of topoisomerase II, within living K562 cancer cells have been studied using surface-enhanced Raman (SER) spectroscopy. The work is based on data of the previously performed model SER experiments dealing with amsacrine/ DNA, drug/topoisomerase II and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. The SER data indicated two kinds of amsacrine interactions in the model complexes with topoisomerase II alone or within ternary complex: non-specific (via the acridine moiety) and specific to the enzyme conformation (via the side chain of the drug). These two types of interactions have been both revealed by the micro-SER spectra of amsacrine within living K562 cancer cells. Our data suppose the specific interactions of amsacrine with topoisomerase II via the side chain of the drug (particular feature of the drug/topoisomerase II and ternary complexes) to be crucial for its inhibitory activity.

The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2+ variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2+ concentrations (up to 10 mM) induced increases in intracellular Mg2+ concentration [Mg2+]i. From a cytosolic basal concentration of 0.8 +/- 0.3 mM in a medium free of Mg2+, an increase in extracellular Mg2+ concentration from 1 to 10 mM, increased cytosolic [Mg2+]i from 1.4 +/- 0.6 to 1.8 +/- 0.8 mM after 10 min (p < 0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2+]i is distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2+ stores involved. The influx pathway for Mg2+ in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2+]i.

An FT-Raman and FT-SERS microprobe with the spatial resolution on the micrometer scale has been developed. The main interfacing components are discussed and the whole setup is validated with the use of different SERS-active substrates: silver and gold colloids and gold island films. Micro-FT-SERS spectra of crocetin, mitoxantrone, and mitoxantrone/DNA complexes have been obtained, and the mass detection limits are found to be on the order of 5 × 102 molecules. Adsorption on the SERS-active substrates does not induce any detectable changes in the all-trans configuration of the crocetin. Adsorption of the mitoxantrone/DNA complex does not induce detectable perturbations of the molecular interactions within the complex. Moreover, interactions between the drug and DNA induces very similar effects in both the resonance Raman and FT-SERS spectra of the drugs. These effects were found to be consistent with the model of mitoxantrone/DNA intercalation proposed from nuclear magnetic resonance and resonance Raman data. The signal-to-noise ratios found indicate that submonolayer amounts of intracellularly localized drugs totaling less than 10−18 mole can be detected by means of the FT-SERS microprobe. Hence, both the extra-low mass detection limit of the technique and its sensitivity to interactions within the supramolecular complexes will in the future allow the drug to be followed within the living cell.

Fourier transform surface enhanced Raman scattering (FT SERS) coupled with a microscope has been used as a probe to obtain information on the interaction of a drug and of its complex with DNA. Micro-FT SERS spectra of the antitumour agent doxorubicin (DOX) at 10 −5 M and of this complex with DNA have been recorded in aqueous silver hydrosol and compared with the corresponding resonance Raman (RR) and SERS spectra at concentrations of 5 × 10 −4 M and 5 × 10 −8 M, respectively. The interactions between the drug and calf thymus DNA induced identical effects in the RR and visible SERS spectra. The data show that the adsorption of the drug-DNA complex on the silver hydrosol does not induce detectable perturbations of the molecular interactions within the complex. Micro-FT SERS spectra were found to be partially different from those obtained in visible SERS spectra. These differences concern the relative enhancement of some vibrational modes which could hardly be observed when resonance excitation was used. The FT SERS approach enables further information to be obtained and additional details on the geometry of the drug-DNA interaction to be revealed. An analysis of the FT SERS spectra of the drug and of its complex with DNA not only confirms the model of interaction proposed using RR and SERS data in the visible, but brings about new information, especially on the vibrations of ring A of the molecule, which are usually masked by the vibrations of rings B and C dominant in the visible SERS spectra.

Molecular interactions of a potent DNA-topoisomerase II (Topo II) inhibitor, m-AMSA [4′-(9-acridinylamino)methanesulphon-m-anisidide] and of its less active isomer o-AMSA in complexes with plasmid DNA, Topo II and Topo II-mediated ternary cleavable complexes were studied by means of surface-enhanced Raman scattering (SERS) spectroscopy. Models for the participation of these drugs in the complexes were proposed according to the analysis of the main vibrational modes of the acridine chromophores in the SERS and resonance Raman (RR) spectra of m-AMSA in comparison with the structurally close derivatives o-AMSA and 9-aminoacridine (9AA). It was found that, under the conditions used, the adsorption on the silver colloid does not perturb the Raman spectra of acridine chromophores. The SERS data indicate the intercalation of the planar acridine moiety within DNA for 9AA and for both o-AMSA and m-AMSA, without pronounced differences for the meta and ortho isomers. The DNA intercalation is likely to involve a μ-μ interaction between the acridine and DNA base pairs, but without contacts via the nitrogen of the acridine ring. This is because the spectral changes observed in the drug-DNA complexes of m-and o-AMSA are different from those observed on acridine deprotonation. In fact, it is the NH group of the side-chain of the drug that we propose to interact with the negatively charged phosphates or edges of DNA and stabilizes the arrangement of the external anilino ring in the minor groove of DNA. The DNA intercalation seems to be present for the acridine moiety of both isomers in the ternary cleavable complexes. However, in the latter the side-chain of m-AMSA, but not of o-AMSA, has been found additionally to participate in specific (enzymatic activity-dependent) interactions with Topo II. The SERS data indicate the ability of m-AMSA to interact specifically with the enzyme alone. The specific m-AMSA-Topo II interactions on formation of the cleavable ternary complex and/or directly with the enzyme should play a key role in the Topo II inhibition and therefore in the anti-tumour activity of the drug.

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident: cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85 +/- 15 nM, HNE (1 microM) produces a [Ca2+]i increase of 91 +/- 66 nM in about 50% of responding cells. At lower concentrations of HNE (0.1 microM, 0.01 microM), the [Ca2+]i rise remains similar, but only 30-40% of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

A new instrument was developed permitting confocal spectral imaging technique to be carried out with a lateral resolution ∼0.3 μm and an axial resolution ∼1.0 μm for specimen areas ranging from 5×5 to 150×150 μm. The modular Raman/fluorescence spectrometer was equipped with a CCD camera, microscope, motorized sample stage, and the confocal entrance chamber. A system of galvanometer controlled mirror scanners equipped the confocal entrance chamber, allowing spectra to be accumulated up to several hundreds of points of sample in parallel, with adjustable spectral and spatial resolution. Stray light rejection property of the Raman spectrometer provides the possibility of the Raman spectral image to be recorded in the low frequency domain. A software was developed to control image accumulation, creation, and treatment. The methods of spectral analysis being applied to a multidimensional set of spectra permit the multiform spectral images to be created. In order to create these images, the different spectral p...

Pre-resonance Raman spectroscopy has been applied to compare the vibrational modes of the R and S chiral isomers of 1-deaza-7,8-dihydropteridine when they are bound to tubulin. The main Raman bands are due to the chromophore and are coupled with the π-π ∗ electronic transition of CC and CN vibrational stretching. On binding to tubulin, the Raman spectra of both isomers are modified. However, the modifications induced are different for each isomer. The Raman bands due to C‖ stretching from the phenyl ring are more strongly modified for the bound R isomer than for the S isomer. This leads us to suggest that R and S isomers differ in terms of their orientation in front of the binding locus of tubulin. In fact, with respect to the orientation of the bulky methyl group, the chromophore of the R isomer is more likely to be positioned against the external surface of either tubulin or GTPase proteins, while that of the S isomer is likely to be positioned away from the surface. The conformational changes induced in tubulin by R and S isomers have also been studied by Fourier transform infrared spectroscopy and by the analysis of amide I and II absorption bands. Both enantiomers induce similar minor changes to the tubulin secondary structure, corresponding to a decrease in the disordered α-helical content and accompanied by an increase in the undefined conformation content.

Fourier transform infrared spectroscopy was used to investigate, without any destructive interference, interactions of the cryoprotective agents, glycerol and dimethyl sulfoxide with Bradyrhizobium japonicum. The intracellular spectrum of glycerol, obtained by subtracting the spectrum of control bacteria from that of glycerol-treated bacteria, showed no differences in infrared features compared to that of pure glycerol. This was not the case when dimethyl sulfoxide treatment was used. The intracellular spectrum displayed important modifications compared to that of the pure chemical. Spectral analysis showed that glycerol and dimethyl sulfoxide uptake required only a few minutes. Moreover, the infrared features resulting from the presence of pure glycerol, in bacteria, were still existent for up to 2 or 3 hours after thawing process. The deconvoluted Amide I and Amide II bands of bacterial proteins reflected important changes in secondary structure after treatment of bacteria with dimethyl sulfoxide (increase in β-sheet and loss of random coil content). In contrast to glycerol, the cryoprotective action of dimethyl sulfoxide probably involves interactions with a wide range of intrabacterial species. © 1995 John Wiley & Sons, Inc.

Surface-enhanced Raman spectra (SERS at Creighton colloidal silver) and UV/visible spectra have been recorded for an antimicrobial agent (pefloxacin) at a biologically active concentration (ca. 10−6 mol/L−1). The adsorption of pefloxacin on the silver surface occurs both via the carboxylate group and the carbonyl of the pyridinone ring. The conjugated part of the molecule is tilted and gives rise to a charge transfer between the drug and the plasmon surface. However the orientation of the drug on the colloid varies with the concentration of pefloxacin, salt addition, and pH. Adsorption via only the carboxylate is privileged in presence of strongly competitive anions such as C1−. Thus the carbonyl of the pyridinone ring is desorbed, and the charge transfer is not detected. In basic medium the competitive OH− ion leads to similar orientation changes. For acidic pHs few residual molecules having a carboxylate function, or few carboxylic species bonded via the pyridinone CO group, remain adsorbed on the aggregated and unstable silver surface. The spectroscopic analyses and the measurements of the particle size of the colloid show that added salt increases the aggregation and enhances the pefloxacin SERS signals if the anion is not competitive. In the presence of NaNO3 an increase of the plasmon oscillations of the metal and a larger number of adsorption sites could explain the SERS amplitude. The competitive anions C1− and OH− to a lesser extent limit the colloid aggregation, pefloxacin adsorption, and SERS enhancement. The influence of the charge transfer on the Raman intensity appears to be weak. © 1995 John Wiley & Sons, Inc.

Innovative diagnostic methods are the need of the hour that could complement conventional histopathology for cancer diagnosis. In this perspective, we propose a new concept based on spectral histopathology, using IR spectral micro-imaging, directly applied to paraffinized colon tissue array stabilized in an agarose matrix without any chemical pre-treatment. In order to correct spectral interferences from paraffin and agarose, a mathematical procedure is implemented. The corrected spectral images are then processed by a multivariate clustering method to automatically recover, on the basis of their intrinsic molecular composition, the main histological classes of the normal and the tumoral colon tissue. The spectral signatures from different histological classes of the colonic tissues are analyzed using statistical methods (Kruskal-Wallis test and principal component analysis) to identify the most discriminant IR features. These features allow characterizing some of the biomolecular alterations associated with malignancy. Thus, via a single analysis, in a label-free and nondestructive manner, main changes associated with nucleotide, carbohydrates, and collagen features can be identified simultaneously between the compared normal and the cancerous tissues. The present study demonstrates the potential of IR spectral imaging as a complementary modern tool, to conventional histopathology, for an objective cancer diagnosis directly from paraffin-embedded tissue arrays.

The present paper provides a spectral comparison between abdominal human skin (Transkin) and pig ear skin using confocal Raman microspectroscopy at 660 nm. Pig ear skin is usually utilized as a substitute for human skin for active ingredients assessment in dermatological and cosmetics fields. Herein, the comparison is made at the level of the stratum corneum (SC), the SC/epidermis junction and the viable epidermis. The 660 nm excitation source appears to be the most appropriate wavelength for such skin characterization. From Raman signatures of both skin types, a tentative assignment of vibrations was performed in the fingerprint and the high wavenumber spectral regions. Significant differences were highlighted for lipid content in in-depth spectra and for hyaluronic acid (HA) and carotenoid in SC spectra. Marked tissular variability was also revealed by certain Raman vibrations. These intrinsic molecular data probed by confocal Raman microspectroscopy have to be considered for further applications such as cutaneous drug permeation.

Complementary diagnostic methods to conventional histopathology are under scrutiny for various types of cancers for rapid and molecular level diagnostics. In this perspective, a biophotonic approach based on infrared spectral micro-imaging combined with multivariate statistical analysis has been implemented on colon tissues. The ability of infrared imaging to investigate the intrinsic biochemical features of cells and tissues has been exploited to develop a new concept of spectral bar coding. To implement this concept, 10 frozen colon tissue samples (five nontumoral and tumoral pairs from five patients) were imaged using infrared spectral micro-imaging in a nondestructive manner. The spectral images were processed by a multivariate clustering method to identify the histological organization in a label-free manner. Spectral information from the epithelial components was then automatically recovered on the basis of their intrinsic biochemical composition, and compared using a statistical method (Mann–Whitney U-test) to construct spectral barcodes specific to each patient. The spectral barcodes representing the discriminant infrared spectral wavenumbers (900–1,800 cm−1) enabled characterization of some of the malignancy-associated biochemical alterations associated with mucin, nucleotides, carbohydrates, and protein regions. This approach not only allowed the identification of common biochemical alterations among all the colon cancer patients, but also revealed a difference of gradient within individual patients. This new concept of spectral bar coding gives insight into the potential of infrared spectral micro-imaging as a complementary diagnostic tool to conventional histopathology, for biochemical level understanding of malignancy in colon cancers in an objective and label-free manner. © 2013 International Society for Advancement of Cytometry

Vibrational spectroscopies (VS), INFRARED SPECTROSCOPY and RAMAN SPECTROSCOPY, are well-established techniques for exploring the chemical composition of samples. VS are based on the molecular vibrations and give a spectral signature also called "molecular fingerprint" characteristic of the studied material. Recent advances in these techniques have rendered them faster, more sensitive, and easier to use. This chapter describes their application to characterize the main glycosaminoglycans-without any sample destruction or degradation. Nowadays, the use of multivariate statistical analysis for analyzing spectral data allows to extract rapidly the discriminant spectral information from large data sets. The combination of VS and this type of data analysis is also discussed in this chapter.

Raman microimaging is a potential analytical technique in health field and presents many possible pharmaceutical applications. In this study, we tested a micrometer spatial resolution probe coupled to a portable Raman imager via an indexed multifiber bundle. At the level of the probe, the fibers were arranged in a circular geometry in order to fit to the pupil of an objective. The imaging potential of this Raman system was assessed on pharmaceutical-like pellets. We showed that this setup permits to record, nearly in real time, Raman images with a micrometer resolution. The collected images revealed a marked heterogeneity in chemicals distribution. Further investigations will be led on cells and biological tissues to evaluate the potential of this Raman imaging device for biomedical applications.

The structure of dimethylcrocetin (DMCRT), prepared by alkaline hydrolysis in methanol of the glucosidic carotenoids extracted from the stigmata of theCrocus sativus L. flowers, has been determined. The molecule has the all-trans configuration and is planar forming a long conjugated system. The C−O bond length values of the two ester groups are shortened as expected. The compound (C11H14O2)2 crystallizes in the orthorhombic space group Pbcn witha=12.5907(7),b=7.5639(5) andc=21.963(2)A. Dimethylcrocetin has characteristic Infrared absorptions at 1697 cm−1 ϑ (C=O) and 1229 cm−1 ϑ (C−O) and characteristic Raman vibrational modes at 1542 cm−1 ϑ (C=C) and 1166 cm−1 ϑ (C−C).

An approach to the determination of secondary structure content in proteins in aqueous solutions based on attenuated total reflection (ATR) Fourier transform infrared (FT-IR) spectrometry is proposed. ATR-FT-IR spectra of eleven proteins with known crystal structures were recorded. An algorithm for careful subtraction of the solvent background was developed and the reproducibility of the spectra was established for a wide range of protein concentrations in aqueous solutions. Two techniques were compared for the determination of secondary structure content [classical least-squares analysis (CLS) and partial least-squares analysis (PLS)] and optimum conditions for their utilization were suggested. The best correlation between the ATR-FT-AIR approach and X-ray diffraction data was obtained with the PLS analysis and the distinction of four types of secondary structures (ordered and disordered α-helix, β-sheet and undefined conformation). The averages of the differences in the percentage content between X-ray and IR secondary structures predicted in the ATR mode are 7.1% and 2.8% for the ordered and disordered α-helix, respectively, 6.5% for the β-sheet and 4.7% for the undefined structure.

A multi-purpose instrument which allows the recording of confocal micro-Raman, micro-SERS and micro-fluorescence spectral images of sample areas from 5 × 5 to l50 × l50 μm with a lateral resolution of ca. 0 3 μm and an axial resolution of ca. 1 μm was developed. The instrument is a combination of two spectrometers both coupled to the same microscope, motorized sample stage, confocal entrance chamber, macro-sample chamber and CCD detector. The first spectrometer includes a double monochromator coupled with a spectrograph and exhibits the properties of a typical high-resolution Raman instrument permitting measurements of the low-wavenumber regions of the spectra. The second spectrometer includes a Notch filter and a spectrograph equipped with two interchangeable low-dispersion gratings and exhibits the properties of a high-luminosity spectrometer, suitable for low resolution, over a wide spectral range and highly sensitive micro-Raman and microfluorescence measurements. The choice of spectrometer most suitable for a particular application can be made automatically without additional prealignment of the system. The system of optical scanners operating in the confocal mode and two-dimensional CCD detection allow the accumulation of spectra from hundreds of points of the sample under the microscope simultaneously. A computer-controlled scanning sample stage and a system using a ‘scanning line’ of the laser beam allow fast recording of well resolved confocal spectral images (CSI) without sample degradation. Conventional images of species could be recorded with a TV–CCD camera through the microscope optics. The software supports all stages of CSI recording and allows the combined treatment of conventional and spectral images including their spatial calibration and conversion of spectral image into a conventional image, to assign point-by-point the spectral data to the conventional image. The applicability of the instrumentation and techniques to the study of polymeric materials, industrial samples and fluid inclusions in minerals was demonstrated. Spectral images based on the micro-SERS analysis of an antitumour drug adsorbed on the hydrosol were recorded and are discussed in terms of their application to the micro-SERS imaging studies of living cells.

Fourier-transform infrared spectroscopy was used to explore structural changes in bacteria under different incubation conditions. In particular, differences between Bradyrhizobium japonicum (BRJ) grown in liquid and on solid media were investigated, as well as the rearrangement of BRJ after transfer from one medium to the other. The FT-IR absorption bands located between 1200 and 900 cm-1 region, vary in spectral shape and intensity when BRJ were suspended in solution medium or plated on solid medium. In agreement with the electronic micrograph data, these spectroscopic changes are due to the changes involving the bacterial wall (peptidoglycan) when BRJ are plated in agar medium. By means of this FT-IR ultrastructural study of Bradyrhizobium japonicum bacteria, it has been possible to follow and to evaluate the rate of the molecular change in bacteria without any destructive interference. This indicates that FT-IR spectroscopy can prove to be a valuable technique in the monitoring of metabolic events in bacterial cells relevant to agriculture as well as environmental and health sciences.

Microcapsuies were prepared from human serum albumin (HSA) through interfacial cross-linking with terephthaloyl chloride (TC). Reaction times were increased from 2 to 60 min, while pH (9.8) and TC concentration (2.5 % w/v) were kept constant. Fourier-transform infrared (FT-IR) spectra of lyophilized microcapsuies were compared. Correlations were established with microcapsuie morphology and size, as had been done in a previous study exploring the effect of increasing pH values. Microcapsuies obtained after 2 min had to be considered separately. minor alterations were observed in the spectrum as compared with pure HSA. They consisted of a decrease of the ester- assigned 1724-crrr1 band and of the carboxylate-assigned 1394-cnrr1 band, attributed to a removal of contaminating lipids of HSA, and an increase of the 1624-cnr1 band, attributed to interchain H bonding following acylation of the NH2 groups. Prolonging the reaction time resulted in a progressive increase of the bands at 1724 (esters), 1795 (anhydrides), and 1624 cm-1 (/J-sheet), in a further decrease of the 1394-cnr1 band (carboxylates), and in marked alterations of the 1340- 1080-crrr’ region. These important changes, which appeared after 5 min, reflect the progressive acylation of the hydroxy and carboxylate groups of HSA. As in the previous series of pH-based assays, important spectral changes were shown to correspond to a decrease in microcapsuie mean size (from 32 to

The first part of this review is devoted to medical applications of Raman spectroscopy as a diagnostic or analytical tool. Studies of human arteries, ocular lenses, living cells and chromosomes are reviewed, in addition to recent advantages in cancer diagnostics using Raman spectroscopy. The second and major part is devoted to a relatively new field, surface-enhanced Raman scattering (SERS) spectroscopy of biomedical species. The SERS effect is accompanied by strong quenching of fluorescence and so enabes the range of species that can be investigated by Raman technique to be extended. The ultra-high sensitivity of SERS enables spectra to be obtained at concentrations down to 10−10 M. A wide range of experiments designed to probe the structure, topology and composition of biomedical species using SERS spectroscopy can be envisioned. Some of these currently being studied are: the determination of the distribution of drugs within a living cell and on the cellular membrane, the selective study of cell membrane components, the analysis of crude biomedical mixtures and extracts and new techniques, based on SERS spectroscopy, Fourier transform SERS spectroscopy and the SERS microprobe method.

Vibrational spectroscopies (VS), INFRARED SPECTROSCOPY and RAMAN SPECTROSCOPY, are well-established techniques for exploring the chemical composition of samples. VS are based on the molecular vibrations and give a spectral signature also called "molecular fingerprint" characteristic of the studied material. Recent advances in these techniques have rendered them faster, more sensitive, and easier to use. This chapter describes their application to characterize the main glycosaminoglycans-without any sample destruction or degradation. Nowadays, the use of multivariate statistical analysis for analyzing spectral data allows to extract rapidly the discriminant spectral information from large data sets. The combination of VS and this type of data analysis is also discussed in this chapter.

Aims: Classical biochemical and molecular methods for discerning cells with epigenetic modifications are often biologically perturbing or even destructive. We wondered whether the noninvasive laser tweezer Raman spectroscopy technique allowed the discrimination of single living human cells undergoing epigenetic modifications. Materials & methods: Human Jurkat leukemic cells were treated with inhibitors of histone deacetylases (trichostatin A and MS-275). Epigenetic changes were monitored through histone electrophoresis, nuclear image cytometry and laser tweezer Raman spectroscopy. Results: Treatment of Jurkat cells with histone deacetylase inhibitors increased histone acetylation and induced chromatin organization changes. Characteristic vibrations, issued from laser tweezer Raman spectroscopy analyses, mostly assigned to DNA and proteins allowed discerning histone deacetylase inhibitor-treated cells from control with high confidence. Statistical processing of laser tweezer Raman spectroscopy data led to the definition of specific biomolecular fingerprints of each cell group. Conclusion: This original study shows that laser tweezer Raman spectroscopy is a label-free rapid tool to identify living cells that underwent epigenetic changes.

This study aims to develop a new FT–IR spectral imaging of tumoral tissue permitting a better characterization of tumor heterogeneity and tumor/surrounding tissue interface. Infrared (IR) data were acquired on 13 biopsies of paraffin-embedded human skin carcinomas. Our approach relies on an innovative fuzzy C-means (FCM)-based clustering algorithm, allowing the automatic and simultaneous estimation of the optimal FCM parameters (number of clusters K and fuzziness index m). FCM seems more suitable than classical ‘hard’ clusterings, as it permits the assignment of each IR spectrum to every cluster with a specific membership value. This characteristic allows differentiating the nuances in the assignment of pixels, particularly those corresponding to tumoral tissue and those located at the tumor/peritumoral tissue interface. FCM images permit to highlight a marked heterogeneity within the tumor and characterize the interconnection between tissular structures. For the infiltrative tumors, a progressive gradient in the membership values of the pixels of the invasive front was also revealed.

Identifying cell response to a chemotherapy drug treatment, in particular at the single cell level, is an important issue in patient management. This study aims at evaluating the effect of gemcitabine on single living cells using micro-Raman imaging. We used as a model the non-small lung cancer cell line, Calu-1, exposed to cytostatic doses (1 nM to 1 mu M for 24 h and 48 h) of gemcitabine, an antitumor drug currently used in the treatment of lung cancer. Following drug treatment as a function of doses and incubation times, the Raman maps of single living cells were acquired. Cell biomolecules (DNA, RNA, and proteins) were chemically extracted and their spectral signatures used to evaluate their respective distribution in the cellular spectral information of control and treated cells. The quantification of these distributions reveals a significant effect of 100 nM gemcitabine at 48 h incubation (concomitant decrease of nucleic acids and increase of proteins). PCA analyses performed both on nuclear and extracted biomolecules spectra show a time-dependent effect of the drug. These promising results reveal that effects of subtoxic doses can be monitored at the single cell level highlighting the importance of such studies for clinical applications.

This study describes a complementary infrared (IR) and Raman spectroscopic investigations of a set of biomolecule representatives of glycosaminoglycans (GAGs) class. Both IR and Raman data exhibit characteristic spectral signatures that allow a direct molecular distinction of these compounds. Comparison of these molecular signatures clearly evidences the differences between heparan sulfate and heparin by computing the intensity ratio between the 1248 cm −1 and 1043 cm −1 peaks, corresponding respectively to sulfate and C─O─C linkages. Identically, chondroitin-4-sulfate and chondroitin-6-sulfate are differentiated via the 730 cm −1 and 853 cm −1 bands (axial orientation) and the 822 cm −1 and 1000 cm −1 bands (equatorial orientation). These orientations concern the sulfate groups of these molecules. Secondly, multivariate statistical methods were employed to analyze the data set. Hierarchical cluster analysis (HCA) of both IR and Raman spectra showed a very good differentiation between the different structures. In addition, the sulfatation degree could be obtained from this classification. Principal component analysis using three principal components (PCs) confirmed the findings of HCA and, furthermore, comparisons of the PC loadings highlight the main molecular differences between the members of this class of biological molecules. It is expected that these microspectroscopic methods will be useful in identifying GAG spectral signatures in complex biological systems.

Surface-enhanced Raman scattering (SERS) spectroscopy is now a well-established phenomenon, which has been thoroughly characterized in a variety of interfacial and colloidal environments. Although some quantitative aspects of the underlying enhancement mechanisms apparently remain unresolved, attention is now shifting towards application of SERS to explore phenomena of chemical, physical, biological and industrial significance. The goal of this review is to appreciate the industrial value of innovative SERS technique on the basis of our experience in development of new SERS-active substrates and in their biomedical and biotechnological applications. Examples of diverse SERS analytical applications as well as some very recent facilities, as SERS microprobe analysis, SERS fiber optics probes, FT-SERS spectroscopy, SERS detection for high-performance liquid chromatography, etc. , are also discussed.

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement.

Background & Aims To follow the local tissue delivery of doxorubicin in HCC explants from patients embolized with drug-eluting beads and to compare it with histologic modifications. Methods Six patients with HCC underwent chemoembolization with doxorubicin-eluting beads (caliber 100–300μm, dose 75–150mg) followed by liver transplantation at different time points (8h to 36days). On sections of the explanted liver, the tissue concentration of doxorubicin was determined radially around bead-occluded vessels with microspectrofluorimetry. The intra/peritumoral location of the beads and the modifications of the surrounding tissue were determined on an adjacent hematein–eosin–saffron-stained section and compared to drug measurements. Results Doxorubicin was detected in the tissue surrounding the beads at all times of explantation. The drug impregnates an area of at least 1.2mm in diameter around the occluded vessel. The tissue concentration of drug ranges from 5μM at 8h to 0.65μM at 1 month. In patient transplanted at 8h, no major tissue modification was observed and we found 42% of the beads occluding intratumoral vessels. Drug concentration was not different around intratumoral and peritumoral occluded vessels. After 9–14days, necrosis was present around 37% of vessels and at 32–36days, around 40% of vessels. Necrotic tissue was associated with a deeper penetration and a higher concentration of the drug than non necrotized areas, though statistically significant only at 32–36days. Conclusions Doxorubicin-eluting beads provide a sustained delivery of drug for a period of 1 month and local tissue concentrations above cytotoxic threshold in HCC-bearing livers.

The outermost layer of the mammalian skin, the stratum corneum (SC), represents the main skin barrier. The SC lipids have a very exceptional composition, as the main lipid classes are ceramides (CER), long-chain fatty acids and cholesterol. Information on the function of each CER subclass and on the relation between CER lipid organisation and composition is of great importance to unravel the mechanism controlling the skin barrier function. Raman spectroscopy has been increasingly used for the study of intra- and inter-molecular structures of long-chain lipid compounds. In this study, we employed Raman spectroscopy to evaluate the effect of (1) the chain length and (2) the polar head architecture on the conformational order and organisational behaviour of CERs. The relation between the structure and the stability of the organisation was studied by monitoring the thermotropic response of each CER in the temperature range between 25 and 95 °C. This work enabled the determination of a correlation between the gauche/trans ratio in the νCC region and the state of the lateral packing. Moreover, it was shown that –OH groups in the α position of the fatty acids reduce the stability while long alkyl chains reinforces the intra- and inter-chains order.

During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers.Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set.IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Raman spectroscopy has proven its potential for the analysis of cell constituents and processes. However, sample preparation methods compatible with clinical practice must be implemented for collection of accurate spectral information. This study aims at assessing, using micro-Raman imaging, the effects of some routinely used fixation methods such as formalin-fixation, formalin-fixation/air drying, cytocentrifugation, and air drying on intracellular spectral information. Data were compared with those acquired from single living cells. In parallel to these spectral information, cell morphological modifications that accompany sample preparation were compared. Spectral images of isolated cells were first analyzed in an unsupervised way using hierarchical cluster analysis (HCA), which allowed delimitation of the cellular compartments. The resulting nuclei cluster centers were compared and revealed at the molecular level that fixation induced changes in spectral information assigned to nucleic acids and proteins. In a second approach, a supervised fitting procedure using model spectra of DNA, RNA, and proteins, chemically extracted from living cells, revealed very small modifications at the level of the localization and quantification of these macromolecules. Finally, HCA and principal components analysis (PCA) performed on individual spectra randomly selected from the nuclear regions showed that formalin-fixation and cytocentrifugation are sample preparation methods that have little impact on the biochemical information as compared to living conditions. Any step involving cell air drying seems to accentuate the spectral deviations from the other preparation methods. It is therefore important in a future context of spectral cytology to take into account these variations.

The in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level. Figure Chemical preservation of cells for Raman microspectroscopy is shown to be strongly dependent on the cell type and the fixative used, in a variety of cell lines, with formalin fixation show to result in spectral content most comparable to that in the live cell

Basal cell carcinoma (BCC) can sometimes lead, through a possible invasion of the dermis and the subcutaneous tissue, to serious local damage to the patient. Several histological types of BCC are reported, among them, the superficial, nodular and infiltrative forms. This study reports the use of polarized Raman microspectroscopy on the nodular and superficial types to discriminate between healthy epidermis and tumor, and between normal and peritumoral stroma. This technique probes additional information than conventional Raman spectroscopy because it is sensitive to the molecular ordering of tissue components. Depolarization ratios and hierarchical cluster analysis demonstrate that polarized Raman microspectroscopy can better identify the tumor and the peritumoral dermis than conventional Raman microspectroscopy, and hence gives potential complementary data about their molecular characteristics (molecular composition, secondary structure of proteins, intra- and/or inter-molecular bonding). Our findings also show that although superficial and nodular types of BCC were analysed, clear differences between the spectra of peritumoral and normal dermis could be detected.

We have investigated the spatial distribution of molecular changes associated with C6 glioma progression using Fourier transform infrared (FT-IR) microspectro-imaging in order to determine spectroscopic markers for early diagnosis of tumor growth. Our results showed that at day 7 after tumor implantation, FTIR investigations displayed a very small abnormal zone associated with the proliferation of C6 cells in the caudate putamen. From this day, rats developed solid and well-circumscribed tumors and invasive areas. The volume of peritumoral areas increased rapidly until day 19. The maturation of the tumor was accompanied by a diminution in its proliferative and invasive area. The presence of necrotic areas was visible from day 15. A non-negative least-squares algorithm was used to quantify spatial distribution of molecular changes in tissues (lipids, nucleic acids, and proteins) associated with glioma progression. Compared to those in normal brain, statistical tests on fit coefficients showed that the concentrations of sphingomyelin (SMY), nucleic acids, phosphatidylserine (PS), and galactocerebroside (GalC) were significantly affected during C6 glioma development. These constituents can be used as spectroscopic markers for C6 glioma progression. Indeed, the concentration of DNA decreased significantly from tumor to invasion, to normal brain tissues, the necrotic area has higher concentrations of the Galc than other areas. The PS content was significantly higher in the peritumoral zone and decreased in the tumor zones matter.

The barrier function of the stratum corneum (SC) is directly related to: (1) the nature and the composition of the lipids in the intercellular spaces and (2) the conformational order of the ceramides within this layer. The aim of this work was to determine Raman descriptors for the lateral packing, the conformation, and the structure of ceramides III, IIIA, and IIIB issued from the same phytosphingosine ceramide and only presenting differences in the number of double bonds in the hydrocarbon chains. Temperature was used as a variable parameter in order to access the different states of the conformational order and supramolecular organization of the three ceramides, and Raman spectra were collected at each temperature. By using a high-resolution Raman spectrometer and working on a spectral range going from 400 to 3,200 cm(-1), we were able to assess simultaneously the different descriptors of structure and organization, i.e., the methyl rocking bands (840-910 cm(-1)) for the chain-end conformers, the C-C skeletal stretching (1,060-1,130 cm(-1)), and the CH stretching region (2,800-3,050 cm(-1)) for the trans and gauche conformations, the CH(2) scissoring bands to follow the changes in the lateral packing, and finally the amide I band to evaluate the state of the H-bonds between the polar and head groups.

Mid-Infrared (IR) micro-spectral imaging is an efficient method to analyze molecular composition of biomedical samples. In clinical oncology, this non-invasive technique is generally used on frozen biopsies to localize and diagnose cancerous tissues in their early stages. However, samples are usually fixed in paraffin in order to be preserved from decay, but the IR signature of paraffin prevents the study of the underlying tissue. To neutralize the paraffin signal from the recorded data, preprocessing methods based on Independent Component Analysis (ICA) and Nonnegatively Constrained Least Squares (NCLS) or on Extended Multiplicative Signal Correction (EMSC) have been recently developed. Then, in order to identify tumor areas, clustering techniques are applied on the preprocessed data, the final result being a false-color map of the biomedical sample which is comparable to the conventional histological image. By allowing each recorded spectrum to be assigned to every cluster, the fuzzy clustering gives more realistic results for unclear tissue boundaries by better highlighting the tumor and peritumoral areas. A recent algorithm based on the redundancy of classes allows to automatically estimate the optimal number of classes and the optimal fuzzy parameter. In this paper, we analyze the effects of the preprocessing methods on the optimal parameter extraction and on the results of the fuzzy clustering on different paraffin embedded cancerous skin samples.