Search

Background Monitoring the transcutaneous permeation of exogenous molecules using conventional techniques generally requires long pre-analytical preparation or labelling of samples. However, Raman spectroscopy is a label-free and non-destructive method which provides spatial distribution of tracked actives in skin. The aim of our study was to prove the interest of Raman imaging coupled with multivariate curve resolution alternating least square (MCR-ALS) analysis in monitoring retinol penetration into frozen and living human skin. Materials and methods After topical treatment of skin samples by free or encapsulated retinol, thin cross sections were analysed by Raman imaging (up to 100 µm depth). Mann-Whitney test was used to identify retinol spectroscopic markers in skin. MCR-ALS was used to estimate retinol contribution in Raman spectral images. Heat maps were constructed to compare the distribution of free and encapsulated retinol in skin models. Results We identified the bands at 1158, 1196 and 1591 cm-1 as specific features for monitoring retinol in skin. Moreover, our MCR-ALS results showed an improvement of retinol penetration (up to 30 µm depth) with the encapsulated form as well as storage reservoir formation in stratum corneum, for each skin model. Finally, greater retinol penetration into living skin was observed. Conclusion This study shows a proof of concept for the evaluation of retinol penetration in skin using Raman imaging coupled with MCR-ALS. This concept needs to be validated on more subjects to include inter-individual variability but also other factors affecting skin permeation (age, sex, pH, etc). Our study can be extended to other actives.

The objective of our research was to investigate the heat-protecting effect of a product ex vivo and in vivo on human hair fibres.A preparatory study was carried out in order to determine an optimal threshold of thermal stress. For this, the structure of cross-sections of the hair fibre was observed by optical microscopy. Then, Scanning Electron Microscopy (SEM) and Confocal Raman Spectroscopy (CRS) were applied to analyse ex vivo and in vivo morphological and molecular damage in hair structure after heat stress. Finally, in vivo tests were used to collect consumer perception.The preparatory study enabled us to determine an optimal stress threshold of 10 heating cycle for SEM and 5 heating cycle for CRS. Based on spectral hierarchical classification using Ward's clustering algorithm, the ex vivo Raman results show that the spectral signature of the hair treated and heated is very close to the negative control. This shows that the product preserves the keratin structure after thermal stress. These results were also confirmed by an in vivo Raman analysis performed on hair samples from 5 donors. In concordance with Raman results, SEM shows that treated hair presents lesser "bubbles" and "crackling" on the hair surface. Finally, the in vivo studies proved that hair was more protected from heat.The authors concluded that the product shows protective properties with respect to morphological and molecular heat damage. We also demonstrate that the product promotes the α-helix keratin conformation and preserves the S-S disulfide bands.L'objectif de notre étude est d'évaluer ex vivo et in vivo l'effet thermoprotecteur d'un produit sur les fibres capillaires humaines. MÉTHODES: Une étude préparatoire a été réalisée afin de déterminer un seuil optimal du stress thermique. Pour cela, la structure des coupes transversales des cheveux a été observée par microscopie optique. Ensuite, la microscopie électronique à balayage (MEB) et la spectroscopie confocale Raman (SCR) ont été appliquées pour analyser les dommages morphologiques et moléculaires (ex vivo et in vivo) de la structure du cheveu après un stress thermique. Enfin, des tests in vivo ont été réalisés pour recueillir la perception des consommateurs. RÉSULTATS: L'étude préparatoire nous a permis de déterminer un seuil de stress thermique optimal correspondant à 10 cycles de chauffage pour la MEB et 5 cycles de chauffage pour la SCR. Basés sur une classification hiérarchique utilisant l’algorithme de Ward, les résultats Raman « ex vivo » montrent que la signature spectrale des cheveux traités et chauffés est très proche du témoin négatif. Cela montre que le produit préserve la structure de la kératine après un stress thermique. Ces résultats ont également été confirmés par une analyse Raman « in vivo » réalisée sur des échantillons de cheveux de 5 donneurs. En concordance avec les résultats Raman, la MEB montre que les cheveux traités présentent moins de « bulles » et de « craquelures » à la surface des cheveux. Enfin, l’étude in vivo a prouvé que les cheveux sont mieux protégés de la chaleur.Les auteurs ont conclu que le produit présente des propriétés protectrices vis-à-vis des dommages thermiques morphologiques et moléculaires. Nous avons démontré également que le produit favorise la conformation de la kératine en hélice-α et préserve les bandes disulfures S-S.

Introduction: Skin aging is the result of many cellular dysfunctions over time particularly the fibroblasts and the keratinocytes. These dysfunctions could be decelerated by the preventive effects of some skin treatments such as Intradermal injections. NCTF 135HA has a polycomponent formulation designed to improve the efficacy of non-cross linked hyaluronic acid (as a micro-filler) on fibroblasts function. Although NCTF 135HA has been used by aesthetic practitioners since 20 years, we have analyzed all in vitro, ex vivo and in vivo studies during past 10 years in order to summarized its anti-aging effect. Methods: In these series of studies, the known effects of HA have been evaluated with this boosted formula. Collagen I synthesis, antiglycation effect and contractile forces developed by fibroblasts were studied. Fibroblasts and keratinocytes proliferations were evaluated in monolayer cells culture. The filling effect thanks to NCTF 135HA injection was ex vivo performed by fringe projection and the protective effect of NCTF 135HA against solar irradiation was performed ex vivo to study proliferation rate, elastin and collagen expression. Two clinical trials were performed on women to evaluate the anti-aging effect of NCTF 135HA injection. Hydration, firmness, radiance, wrinkles size, pore size, thickness and density of the dermis were analyzed. Results: Collagen I, anti-AGE products, cell proliferation and contractile forces were significantly increased with NCTF 135HA in culture medium. Ex vivo studies showed that NCTF 135HA increased, cells proliferation rate, elastin and collagen production. In addition, the filling effect of the NCTF 135HA is significantly superior to placebo. The first clinical trial shows that NCTF 135HA injection significantly increased tonicity, hydration and radiance of the skin and significantly decreased wrinkle score. The second clinical trial demonstrated that NCTF 135HA injection significantly increased dermis density and thickness, skin homogeneity and skin radiance and significantly decreased pore size and wrinkle depth and volume. Conclusion: In vitro, ex vivo and in vivo studies showed that this minimally invasive technique could be used in order to improve the skin quality which could have a visual effect on preventing the aging signs.

Correction for ‘Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin’ by Sana Tfaili et al., Analyst, 2012, 137, 3673–3682, DOI: 10.1039/C2AN16292J.

Objective Although xerosis is a common skin disorder among the population, there is no in vivo global study focusing on xerotic skin. Hence, the objective of this study was to characterize xerotic skin from the surface to the molecular scale with in vivo and non-invasive approaches. Methods For this purpose, 15 healthy volunteers with normal skin and 19 healthy volunteers with xerotic skin were selected by a dermatologist, thanks to a visual scorage. Firstly, the skin surface was characterized with biometric measurements. Then, the state of skin dryness was assessed by in vivo confocal microscopy. The molecular signature of xerotic skin was then determined by in vivo confocal Raman microspectroscopy. Finally, an identification of stratum corneum (SC) lipids was performed using Normal phase liquid chromatography (NP-LC) coupled to two detectors: Corona and High Resolution/Mass Spectroscopy (HR/MS). Results Results obtained at the skin surface displayed an increase in the transepidermal water loss (TEWL) and a decrease in the hydration rate in xerotic skin. Confocal microscopy revealed an alteration of the cell shape in xerotic skin. Moreover, confocal Raman microspectroscopy demonstrated directly in vivo and non-invasively the lack of organization and conformation of lipids in this skin. Finally, HPLC analyses revealed that the three ceramide sub-classes (NdS, NS and EOP) significantly decrease in xerosis. Altogether, these results identify parameters for the characterization of xerotic skin compared to normal. Conclusion This study highlighted discriminative parameters from the surface to the molecular level in vivo and non-invasively between xerotic and normal skins. These results will be useful for the development of new cosmetic active ingredients dedicated to xerotic skin.

Atopic dermatitis (AD) is the most common skin inflammatory disease, affecting up to 3% of adults and 20% of children. Skin barrier impairment is thought to be the primary factor in this disease. Currently, there is no method proposed to monitor non-invasively the different molecular disorders involved in the upper layer of AD skin. Raman microspectroscopy has proved to be a powerful tool to characterize some AD molecular descriptors such as lipid content, global hydration level, filaggrin and its derivatives. Our investigations aimed to extend the use of in vivo Raman microspectroscopy as a rapid and non-invasive diagnostic technique for lipid conformation and organization, protein secondary structure and bound water content analysis in atopic skin. Our approach was based on the analysis of Raman data collected on the stratum corneum (SC) of 11 healthy and 10 mild-to-moderate atopic patients. Atopic skin revealed a modification of lipid organization and conformation in addition to the decrease of the lipid-to-protein ratio. This study also highlighted a reduction of the bound water and an increase in protein organized secondary structure in atopic skin. All these descriptors worsen the barrier function, state and appearance of the skin in AD. This precise and relevant information will allow an in vivo follow-up of the pathology and a better evaluation of the pharmacological activity of therapeutic molecules for the treatment of AD.

Objective In hair care cosmetic products’ evaluation, one commonly used method is to evaluate the hair appearance as a gold standard in order to determine the effect of an active ingredient on the final state of the hair via visual appreciation. Although other techniques have been proposed for a direct analysis of the hair fibres, they give only surface or structural information, without any accurate molecular information. A different approach based on confocal Raman spectroscopy has been proposed for tracking in situ the molecular change in the keratin directly in the human hair fibres. It presents a high molecular specificity to detect chemical interactions between molecules and can provide molecular information at various depths at the cortex and cuticle levels. Methods To evaluate the potential of confocal Raman spectroscopy in testing the efficiency of cosmetic ingredients on keratin structure, we undertook a pilot study on the effectiveness of a smoothing shampoo on natural human hair, by analysing α‐helix and β‐sheet spectral markers in the Amide I band and spectral markers specific to the cystin sulfur content. Results We confirmed that an active proved to be effective on a gold standard decreases α‐helix keratin conformation and promotes β‐sheet keratin conformation in the hair fibres. We also showed that treatment with the effective active decreases the intensity of covalent disulfide (S–S at 510 cm‐1) cross‐linking bands of cysteine. These data confirm that the effective active also acts on the tertiary structure of keratin. Conclusion From these experiments, we concluded that the effective active has a smoothing effect on the human hair fibres by acting on α‐helix and β‐sheet keratin conformation and on the tertiary structure of keratin. Based on these results, confocal Raman spectroscopy can be considered a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres. Moreover, this analytical technique has the advantage of being non‐destructive and label free; in addition, it does not require sample extraction or purification and it can be applied routinely in cosmetic laboratories., Confocal Raman spectroscopy can be considered as a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres.

Skin is one of the most important organs of the human body because of its characteristics and functions. There are many alterations, either pathological or physiological, that can disturb its functioning. However, at present all methods used to investigate skin diseases, non-invasive or invasive, are based on clinical examinations by physicians. Thus, diagnosis, prognosis and therapeutic management rely on the expertise of the practitioner, the quality of the method and the accessibility of distinctive morphological characteristics of each lesion. To overcome the high sensitivity of these parameters, techniques based on more objective criteria must be explored. Vibrational spectroscopy has become as a key technique for tissue analysis in the biomedical research field. Based on a non-destructive light/matter interaction, this tool provides information about specific molecular structure and composition of the analyzed sample, thus relating to its precise physiopathological state and permitting to distinguish lesional from normal tissues. This label-free optical method can be performed directly on the paraffin-embedded tissue sections without chemical dewaxing. In this study, the potential of the infrared microspectroscopy, combined with data classification methods was demonstrated, to characterize at the tissular level different types of inflammatory skin lesions, and this independently from conventional histopathology.

Parmi les cancers de la peau, les deux types de carcinomes cutanes (basocellulaires et spinocellulaires) sont difficilement distinguables lors de l’examen clinique. Or, il est crucial de les differencier car les seconds necessitent un traitement et une surveillance specifiques. Pour reduire au minimum l’intervention humaine, forcement subjective, la micro-imagerie infrarouge presente un potentiel certain lors du diagnostic… a condition de savoir traiter les images des tissus inclus en paraffine qui ont ete preleves. Ce defi a ete releve et est en passe de devenir la prochaine methode de diagnostic totalement objective et predictive.

This study proposes a comparative investigation of different linear and non-linear chemometric methods applied to the same database of infrared spectra for filamentous fungi discrimination and identification. The database was comprised of 277 strains (14 genera, 36 species), identified and validated by DNA sequencing, and analyzed by high-throughput Fourier Transform Infrared (FTIR) spectroscopy in the 4000–400 cm−1 wavenumber range. A cascade of 20 supervised models based on taxonomic ranks was constructed to predict classes until the species taxonomic rank. The cascade modeling was used to test 11 algorithms (5 linear and 6 non-linear) of supervised classification methods. To assess these algorithms, indicators of classification rates and McNemar's tests were defined and applied in the same way to each of them. For non-linear algorithms, the KNN (K Nearest Neighbors) method proved to be the best classifier (78%). Linear algorithms, PLS-DA (Partial Least Square-Discriminant Analysis) and the SVM (Support Vector Machine) showed better performances than non-linear methods with the best classification potential (∼93%). The SVM and PLS-DA were comparable and a possible complementarity between these two algorithms was highlighted.

We have investigated the potential of Raman microspectroscopy combined with supervised classification algorithms to diagnose a blood lymphoproliferative disease, namely chronic lymphocytic leukemia (CLL). This study was conducted directly on human blood smears (27 volunteers and 49 CLL patients) spread on standard glass slides according to a cytological protocol before the staining step. Visible excitation at 532 nm was chosen, instead of near infrared, in order to minimize the glass contribution in the Raman spectra. After Raman measurements, blood smears were stained using the May-Grünwald Giemsa procedure to correlate spectroscopic data classifications with cytological analysis. A first prediction model was built using support vector machines to discriminate between the two main leukocyte subpopulations (lymphocytes and polymorphonuclears) with sensitivity and specificity over 98.5%. The spectral differences between these two classes were associated to higher nucleic acid content in lymphocytes compared to polymorphonuclears. Then, we developed a classification model to discriminate between neoplastic and healthy lymphocyte spectra, with a mean sensitivity and specificity of 88% and 91% respectively. The main molecular differences between healthy and CLL cells were associated with DNA and protein changes. These spectroscopic markers could lead, in the future, to the development of a helpful medical tool for CLL diagnosis.

International audience; This study evaluated the midterm delivery of doxorubicin in liver specimens from patients (N = 4) with hepatocellular carcinoma treated with drug-eluting embolic (DEE) transarterial chemoembolization. The patients had surgical resection 57, 79, 80 and 105 days after doxorubicin DEE chemoembolization. Doxorubicin concentrations inside embolic particles and in surrounding tissues were assessed by infrared microspectroscopy and microspectrofluorimetry, respectively. Embolic particles still contained doxorubicin and provided sustained drug delivery within targeted tissues 80 days after chemoembolization. Doxorubicin was undetectable after 105 days. In addition, aggregation of embolic particles inside vessel lumina was associated with slower doxorubicin elution and higher tissue concentrations when the number of aggregated embolic particles increased.

International audience; This paper presents a procedure that digitally neutralizes the contribution of paraffin to FTIR hyperspectral images. A brief mathematical derivation of the procedure is demonstrated and applied on one normal human colon sample to exemplify the de-waxing procedure. The proposed method includes construction of a paraffin model based on PCA, EMSC normalization and application of two techniques for spectral quality control. We discuss every step in which the researcher needs to take a subjective decision during the de-waxing procedure, and we explain how to make an adequate choice of parameters involved. Application of this procedure to 71 hyperspectral images collected from 55 human colon biopsies (20 normal, 17 ulcerative colitis, and 18 adenocarcinoma) showed that paraffin was appropriately neutralized, which made the de-waxed images adequate for analysis by pattern-recognition techniques such as k-means clustering or PCA-LDA.

Proper hydration of the stratum corneum (SC) is important for maintaining skin's vital functions. Water loss causes development of drying stresses, which can be perceived as 'tightness', and plays an important role in dry skin damage processes. However, molecular structure modifications arising from water loss and the subsequent development of stress has not been established. We investigated the drying stress mechanism by studying, ex vivo, the behaviors of the SC components during water desorption from initially fully hydrated samples using Raman spectroscopy. Simultaneously, we measure the SC mechanical stress with a substrate curvature instrument. Very good correlations of water loss to the mechanical stress of the stratum corneum were obtained, and the latter was found to depend mainly on the unbound water fraction. In addition to that, the water loss is accompanied with an increase of lipids matrix compactness characterized by lower chain freedom, while protein structure showed an increase in amount of α-helices, a decline in α-sheets, and an increase in folding in the tertiary structure of keratin. The drying process of SC involves a complex interplay of water binding, molecular modifications, and mechanical stress. This article provides a better understanding of the molecular mechanism associated to SC mechanics.

Complementary diagnostic methods to conventional histopathology are currently being investigated for developing rapid and objective molecular-level understanding of various disorders, especially cancers. Spectral histopathology using vibrational spectroscopic imaging has been put in the frontline as potentially promising in this regard as it provides a “spectral fingerprint” of the biochemical composition of cells and tissues. In order to ascertain the feasible conditions of vibrational spectroscopic methods for tissue-imaging analysis, vibrational multimodal imaging (infrared transmission, infrared-attenuated total reflection, and Raman imaging) of the same colon tissue has been implemented. The spectral images acquired were subjected to multivariate clustering analysis in order to identify on a molecular level the constituent histological organization of the colon tissue such as the epithelium, connective tissue, etc., by comparing the cluster images with the histological reference images. Based on this study, a comparative analysis of important factors involved in the vibrational multimodal imaging approaches such as image resolution, time constraints, their advantages and limitations, and their applicability to biological tissues has been carried out. Out of the three different vibrational imaging modalities tested, infrared-attenuated total reflection mode of imaging appears to provide a good compromise between the tissue histology and the time constraints in achieving similar image contrast to that of Raman imaging at an approximately 33-fold faster measurement time. The present study demonstrates the advantages, the limitations of the important parameters involved in vibrational multimodal imaging approaches, and their potential application toward imaging of biological tissues.

Histopathology remains the gold standard method for colon cancer diagnosis. Novel complementary approaches for molecular level diagnosis of the disease are need of the hour. Infrared (IR) imaging could be a promising candidate method as it probes the intrinsic chemical bonds present in a tissue, and provides a "spectral fingerprint" of the biochemical composition. To this end, IR spectral histopathology, which combines IR imaging and data processing techniques, was employed on seventy seven paraffinized colon tissue samples (48 tumoral and 29 non-tumoral) in the form of tissue arrays. To avoid chemical deparaffinization, a digital neutralization of the spectral interference of paraffin was implemented. Clustering analysis was used to partition the spectra and construct pseudo-colored images, for assigning spectral clusters to various tissue structures (normal epithelium, malignant epithelium, connective tissue etc.). Based on the clustering results, linear discriminant analysis was then used to construct a stringent prediction model which was applied on samples without a priori histopathological information. The predicted spectral images not only revealed common features representative of the colonic tissue biochemical make-up, but also highlighted additional features like tumor budding and tumor-stroma association in a label-free manner. This novel approach of IR spectral imaging on paraffinized tissues showed 100% sensitivity and allowed detection and differentiation of normal and malignant colonic features based purely on their intrinsic biochemical features. This non-destructive methodology combined with multivariate statistical image analysis appears as a promising tool for colon cancer diagnosis and opens up the way to the concept of numerical spectral histopathology.

Routine identification of fungi based on phenotypic and genotypic methods can be fastidious and time-consuming. In this context, there is a constant need for new approaches allowing the rapid identification of molds. Fourier-transform infrared (FTIR) spectroscopy appears as such an indicated method. The objective of this work was to evaluate the potential of FTIR spectroscopy for an early differentiation and identification of filamentous fungi. One hundred and thirty-one strains identified using DNA sequencing, were analyzed using FTIR spectroscopy of the mycelia obtained after a reduced culture time of 48 h compared to current conventional methods. Partial least square discriminant analysis was used as a chemometric method to analyze the spectral data and for identification of the fungal strains from the phylum to the species level. Calibration models were constructed using 106 strains pertaining to 14 different genera and 32 species and were used to identify 25 fungal strains in a blind manner. Identification levels of 98.97% and 98.77% achieved were correctly assigned to the genus and species levels respectively. FTIR spectroscopy with its high discriminating power and rapidity therefore shows strong promise for routine fungal identification. Upgrading of our database is ongoing to test the technique's robustness.

BACKGROUND: The rabbit Vx2 liver tumor is a fast-growing carcinoma model commonly used to study tumor behaviours under cancer treatments. The reduction of tumor viability and the degree of induced necrosis are the common criteria to evaluate the efficacy of cancer treatments. Currently, it is not easy to perform a rapid and reproducible tissue quantification by histopathological analysis. OBJECTIVE: Our objective was to use infrared-imaging combined with linear-discriminant-analysis model (LDA) to automatically quantify the necrotic effect induced by doxorubicin-eluting-implants (DEI) on a Vx2 liver tumors model. METHODS: Three rabbits were subjected to DEI treatment and compared to a control group (CTRL) of nine rabbits. Tumor bearing livers were resected, fixed in formalin and embedded in paraffin. On two consecutive sections, we performed hematoxylin–eosin–saffron (HES) staining and infrared-imaging. Infrared-images were then subjected to the LDA-model analysis. RESULTS: The LDA and HES images strongly correlated for viable and necrotic tumor tissues. For the DEI group, the model determined that the surface of viable tumor represented 0.24 ± 5% of the tumor (CTRL: 55.71 ± 17%, P< 0.0001) and the necrotic tissue 66.46 ± 20% of the tumor (CTRL: 18.45 ± 12%, P< 0.0001). CONCLUSIONS: Our results showed that infrared-imaging coupled with LDA-model analysis could be a helpful to easily and objectively assess the treatment efficacy.

Background Skin dryness is an omnipresent symptom in various types of skin disorders. Thereby, a large panel of skin care products is developed for therapeutic purposes. However, there is still a lack of non-invasive methods to determine the mechanisms of action of moisturizers at the molecular level. Methods In the present study, confocal Raman spectroscopy coupled to classical least square analyses and ATR-FTIR were used to investigate the effect of different molecules on the stratum corneum (SC) hydration degree and barrier state. First, hygroscopic property was determined by analyzing samples at 90% RH; secondly, the water barrier function was evaluated after the dehydration process (4% RH). The molecules penetration kinetics across SC were also studied for 2 h. Results Using the present approach, glycerin and propylene glycol were found to be humectants; lanoline showed occlusive action, lactic acid has both humectant and barrier enhancer properties, and ethylhexyl palmitate and caprylic/capric acid triglyceride seemed to be emollients. These observations are in accordance with literature. Conclusion The present method non-invasively characterizes the mechanism of action of tested molecules. This may improve knowledge of new molecules' structure–activity relationship and help make an effective therapeutic concept dealing with the various skin dysfunctions.

Skin aging is the most visible sign of aging of the body. This complex process involves molecular and structural alterations of the main skin constituents. The major cutaneous constituent is type I collagen that gives strength and resilience to the skin. This macromolecule possesses a particular triple helix structure and is arranged in the form of a fibrous network. Water plays a crucial role for maintaining this molecular assembly which is altered during intrinsic chronological aging. To investigate some of these structural alterations, Raman microspectroscopy was employed since this biophotonic approach permits to probe the biomolecular composition of the skin in a non-destructive manner. In addition, type I collagen gives specific Raman vibrations; some of which being sensitive to the molecule conformation and to the water environment. In this purpose, Raman spectra of four skin samples of different ages (two samples of 40 year old and two samples of 70 year old) were collected by varying the relative environmental humidity. Our experiments highlighted spectral differences between the four samples. The analysis of the Raman data permitted to suggest a model for the interactions between collagen and water molecules and a decrease in collagen fibers compactness with chronological aging. Our study demonstrates that Raman spectroscopy can be a useful tool to investigate skin aging, with innovative applications in dermatology as well as in cosmetics. Copyright © 2013 John Wiley & Sons, Ltd.

The mechanical properties of the Stratum Corneum (SC) have been studied by different authors at the macroscopic level, but the modification of its ultra structure during mechanical extension remains unknown. Moreover, little is described about the effect of the mechanical stress on SC barrier function. In this study, we have examined the SC structure changes, at the molecular level, during uniaxial tensile experiments. This was performed on isolated SC samples using Raman spectroscopy. We could identify the strain status of the analyzed samples by using combination of Raman spectra and Partial Least Squares processing. In addition, this approach provided information about lipids and proteins behavior during the sample extension. The structure of the intercellular lipids bilayer became less organized up to ~9% deformation. For higher strains, a plateau corresponding to the minimum organization is observed till the complete failure of the sample. In the same time, protein structures including desmosomes, were characterized by monotonic secondary structure modifications for deformations up to ~9% followed by a plateau. These observations are relevantly demonstrating the effect of extension on the skin barrier state. Such an approach could be objectively used for clinical applications to evaluate skin discomfort degree and skin elastic behavior. This could therefore help with proof of efficacy for cosmetic and dermatologic products. Copyright © 2013 John Wiley & Sons, Ltd.

Calcium phosphate coatings are synthesized on titanium alloy (Ti6Al4V) substrates by pulsed electrodeposition. This work aims to observe the morphological modifications of the coating when an amino acid is added to the electrolytic solution used in the process. The effects of two amino acids (glutamic acid and aspartic acid) are studied at a low and a high concentration. The coating morphology is observed at a nanometer scale by field emission gun-scanning electron microscopy (FEG-SEM). The structural characterization of the coating is performed by Fourier transformed infrared spectroscopy (FT-IR), Raman spectroscopy and X-ray diffraction (XRD). Moreover, corrosion measurements of the prosthetic surfaces are carried out by potentiodynamic polarization experiments in a physiological solution named Dulbecco's modified eagle medium (DMEM). The results show that the addition of an amino acid to the electrolytic solution leads to the decrease of the size of the crystallites which compose the prosthetic calcium phosphate coating that becomes denser and less porous than the coatings obtained without amino acid. Consequently, the corrosion behavior of the prosthetic material immersed in DMEM is improved.

Assessing the tumor invasiveness is a paramount diagnostic step to improve the patients care. Infrared spectroscopy access the chemical composition of samples; and in combination with statistical multivariate processing, presents the capacity to highlight subtle molecular alterations associated with malignancy development. Our investigation demonstrated that infrared signatures of cell lines presenting various invasiveness phenotypes contain discriminant spectral features, which are useful informative signals to implement an objective invasiveness scale. This last development reflects the interest of vibrational approach as a candidate biophotonic label-free technique, usable in routine clinics, to characterize quantitatively tumor aggressiveness. In addition, the methodology can reveal the heterogeneity of cancer cells, opening the way to further researches in cancer science.

Confocal Raman microspectroscopy is a relevant and useful tool to perform in vivo diagnosis of cutaneous tissues noninvasively and without labeling. This optical technique provides in-depth molecular and conformational characterization of skin. Unfortunately, spectral distortions occur due to elastic scattering. Our objective is to correct the attenuation of in-depth Raman peaks intensity by considering elastic scattering in biological tissues. In this purpose, a correction model was constructed using skin scattering properties as parameters thus enabling quantitative analysis. The work presented here is a technique of in vivo Diffuse Reflectance Micro-Spectroscopy called Micro-DRS. It achieves optical properties characterization in the skin layers probed by Raman microspectroscopy. The Micro-DRS setup can easily be coupled to a confocal Raman micro-probe to perform simultaneous measurements. Thanks to Monte Carlo simulations and experimental results obtained on homemade solid phantoms mimicking skin optical properties, we show that it is possible to measure the absorption coefficient μ a , the reduced scattering coefficient μ s ', the scattering coefficient μ s and the anisotropy of scattering g with this new apparatus. The measured scattering properties can be used subsequently as parameters in our correction model. Coupled to a Raman micro-spectrometer, Micro-DRS enables a quantitative analysis when tracking drug penetration through skin and it can be used independently to provide additional diagnosing criterions.

The main function of the stratum corneum (SC) is for protection against external aggression. This is described as the barrier function. It mainly depends on the presence of a lipid matrix composed of ceramides, free fatty acids, cholesterol and its derivatives in the intercellular spaces. Previous studies have reported the application of Raman spectroscopy to reveal the organization of SC lipids and the state of their barrier functions. Several spectral features are directly informative about the lateral packing and the conformational order. In this work, in vivo Raman spectroscopy is used to asses the state of the SC lipid content and thus its barrier function, directly on the skin. To study the effect of natural aging on the organization of these lipids, spectra were collected from the internal side of the forearms of twenty volunteers aged from 22 to 64. Multivariate data processing enabled separation of the in vivo spectra according to the volunteers' ages. Spectral signatures show small variations, indicating a slight change in the lateral packing of SC lipids with aging of the skin.

Assessment of liver fibrosis is of paramount importance to guide the therapeutic strategy in patients with chronic hepatitis C (CHC). In this pilot study, we investigated the potential of serum Fourier transform infrared (FTIR) spectroscopy for differentiating CHC patients with extensive hepatic fibrosis from those without fibrosis. Twenty-three serum samples from CHC patients were selected according to the degree of hepatic fibrosis as evaluated by the FibroTest: 12 from patients with no hepatic fibrosis (F0) and 11 from patients with extensive fibrosis (F3-F4). The FTIR spectra (ten per sample) were acquired in the transmission mode and data homogeneity was tested by cluster analysis to exclude outliers. After selection of the most discriminant wavelengths using an ANOVA-based algorithm, the support vector machine (SVM) method was used as a supervised classification model to classify the spectra into two classes of hepatic fibrosis, F0 and F3-F4. Given the small number of samples, a leave-one-out cross-validation algorithm was used. When SVM was applied to all spectra (n = 230), the sensitivity and specificity of the classifier were 90.1% and 100%, respectively. When SVM was applied to the subset of 219 spectra, i.e., excluding the outliers, the sensitivity and specificity of the classifier were 95.2% and 100%, respectively. This pilot study strongly suggests that the serum from CHC patients exhibits infrared spectral characteristics, allowing patients with extensive fibrosis to be differentiated from those with no hepatic fibrosis.

International audience; Using biophotonics techniques to retrieve prognostic intracellular signatures : IHMO project aims at developing a multimodal microscopy platform that includes in a single machine Raman micro spectroscopy and multispectral imaging used for tumor diagnosis and prognosis. Lymphocytes from 24 leukaemic patients suffering from hyper leukocytosis Chronic Lymphoid Leukemia and from 11 healthy individual have been studied, using around 90 cells per blood sample. Blood smears were prepared on microscopy slides; cells were localized by optical microscopy, and Raman microspectroscopy spectra were acquired on cell nuclei. Afterward multi-Z stacks images of each cell were acquired for eight bands distributed in the visible spectrum. Raman data's were classified using a Support Vector Machine algorithm that provided a molecular signature that allowed for distinguishing lymphocytes from other nucleated blood components with 99.6% sensibility and 98.8% specificity. Then the algorithm was used for developing a classification model splitting leukaemic and healthy smears; sensibility was 95% and specificity was 87.5% among the spectra used for evaluation: 1540 from 11 leukaemic patients and 516 from seven healthy individual. IHMO project has demonstrated the power of Raman microspectroscopy for cell classification. Morphological descriptors obtained from multi-Z and multispectral images provide another independent classification that still needs to be assessed. The microscopy platform can be used more generally in the field of cytohaematology, however application to cytological and histological pathology would need further developments.

The calculation of an approximate Simple symmetry Valence Force Field including determination of diagonal symmetry valence force constants is achieved by using an iterative process derived from the Stepwise Coupling Method (SCM) in the form of the Matrix Polynomial Expansion Method (M.P.E.M.) of Alix. The discussion on the method itself and the comparison with similar methods such as the Eigen-Vector Method (EVM) is given in detail. Numerical examples are given to illustrate the effectiveness of the method.

The potential of Raman spectroscopy for ex vivo and in vivo classification of normal and glioblastoma brain tumor development was investigated. High-quality spectra of normal and tumor tissues were obtained using a portable Raman spectrometer coupled to a microprobe with a signal integration time of 5 s. Ex vivo results demonstrated that by using the biochemical information contained in the spectra, we were able to distinguish between normal brain features (white and gray matter), invasion, and tumor tissues with a classification accuracy of 100%. Differences between these features resulted from variations in their lipid signal contributions, which probably reflect differences in the level of myelinization. This finding supports the ability of in vivo Raman spectroscopy to delineate tumor margins during surgery. After implanting C6 cells in rat brain, we monitored, in vivo, the development of glioblastoma tumor from days 0 to 20 post-implantation (PI). The classification exhibited a clear separation of the data into two clusters: one cluster was associated with normal brain tissues (cortex), and the second was related to data measured from tumor evolution. The second cluster could be divided into two subclusters, one associated with tumor tissue from 4 to 13 days PI and the second related to tumor tissue from 15 to 20 days PI. Histological analysis reveals that the differences between these two subclusters are: the presence of a massive infiltration zone in the brain tissue from 4 to 13 days PI, and; a maturation of the tumor characterized by the appearance of edematous and necrotic zones, as well as a diminution in the proliferative and invasive area, from 15 days. This work demonstrates the potential of Raman spectroscopy to provide diagnostic information for the early detection of tumors in vivo.

International audience; The influence of Erythromycin, Roxithromycin, Amoxicillin, Tetracycline and Sulfamethoxazole on municipal sludge in batch reactors was investigated. The study was focused on extracellular polymeric substances (EPS) as indicator of bacteria sensitivity to toxic agents. The EPS were analysed by UV-Vis and FT-IR spectroscopies and by size exclusion chromatography. It was found that Erythromycin and Roxithromycin induced a significant increase of bound EPS in flocs. This was attributed to a protection mechanism of the bacteria. Erythromycin was the only antibiotic which inhibited COD and nitrogen removal.

Travo A, Piot O, Wolthuis R, Gobinet C, Manfait M, Bara J, Forgue-Lafitte M-E & Jeannesson P (2010) Histopathology 56, 921–931 IR spectral imaging of secreted mucus: a promising new tool for the histopathological recognition of human colonic adenocarcinomas Aims: 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. Methods and results: 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. Conclusions: IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Over the last few years, FTIR spectroscopy has become a potential analytical method in tissue and cell studies for cancer diagnosis. This has opened a way towards clinical applications such as a tool that would scan samples to assess the presence or absence of malignant cells in biopsies, or as an aid to help pathologists to better characterise those cells that are suspicious but not diagnostic for cancer. The latter application has the problem that in order to assess these cells pathologists would have already dealt with stained samples. Therefore, it is important to understand how staining would affect the spectra of cells. To this purpose, we have conducted this study in order to clarify, first, how haematoxylin and eosin (H&E) and Papanicolau (Pap) stainings affect the spectra of single cells and, second, whether FTIR spectroscopy could differentiate between stained lung cancer cells and their normal counterparts. Furthermore, different cell preparations (cytospin, and smear) used in cytological diagnosis were assessed. Experiments performed using a bright infrared (IR) source (synchrotron) showed that both H&E and Pap staining induced marked changes in the lipid and amide-II band regions. Despite this, FTIR spectroscopy of already stained cells is capable of differentiating between lung cancer cells and their normal counterparts. The clinical applications of this methodology are discussed.

Purpose To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests. Materials and Methods Left lobe hepatic artery embolization was performed in 10 pigs with 100–300-μm or 700–900-μm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100–300-μm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron–stained sections. Results Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time ( P = .0004). The drug was detected at distances as far as 600 μm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 M to 6.80 M, which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100–300 μm in size induced more necrosis than 700–900-μm beads ( P = .0036). Conclusions Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects.

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.

Ibuprofen loaded embolization beads (IBU-BB) have been developed to reduce inflammation and pain following uterine artery embolization for the treatment of uterine fibroids. The present work has investigated the elution properties of IBU-BB in situ after embolization with Fourier Transform Infrared Microspectroscopy (FTIRMS). Twelve sheep underwent uterine artery embolization with IBU-BB (485 mM) or control unloaded beads. IBU concentration was determined inside the beads and in the tissue surrounding the beads using FTIRMS of uterine tissue sections sampled 24 h or 1 week after embolization. After 24 h, IBU concentration inside the bead was only 18.6 mM out of the 485 mM initially loaded (p0.0001, univariate sign test). The concentration in the tissue around the beads was 8 mM, which is well above the in vitro therapeutic levels (6 microM). After one week the concentration of IBU had decreased to 4.9 mM in the beads (p = 0.0502, Mann Whitney) and no IBU was detected in the surrounding tissue. This work has demonstrated that IBU-BB can provide a sustained release of the anti-inflammatory drug over at least one week. The in vivo elution properties of IBU-BB may be suitable to alleviate pain and inflammation after embolization.

Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

Raman microspectroscopy allows probing subcellular compartments and provides a unique spectral fingerprint indicative of endogenous molecular composition. Although several spectroscopic cell studies have been reported on fixed samples, only few attempts concern single growing cells. Here, we have tested different optical substrates that would best preserve cell integrity and allow direct measurement of Raman spectra at the single living cell level. Calu-1 lung cancer cells were used as a model and their morphology and growth were assessed on Raman substrates like quartz, calcium fluoride, and zinc selenide. Data show that quartz was the most appropriate taking into consideration both cell morphology and proliferation rate (47% on quartz vs. 55% of BrdU-positive cells on conventional plastic). Using quartz, 40 cells were analysed and Raman spectra were collected from nuclei and cytoplasms using a 785 nm laser excitation of 30 mW at the sample, in the spectral range of 580-1750 cm(-1), and an acquisition time of 2 x 10 sec/spectrum. Discriminant spectral information related to nucleus and cytoplasm were extracted by multivariate statistical methods and attributed to nucleic acids, lipids, and proteins. Finally, Raman spectral imaging was performed to show the distribution of these components within the cell.

The purpose of the study was to investigate molecular changes associated with glioma tissues using FT-IR microspectroscopic imaging (FT-IRM). A multivariate statistical analysis allowed one to successfully discriminate between normal, tumoral, peri-tumoral, and necrotic tissue structures. Structural changes were mainly related to qualitative and quantitative changes in lipid content, proteins, and nucleic acids that can be used as spectroscopic markers for this pathology. We have developed a spectroscopic model of glioma to quantify these chemical changes. The model constructed includes individual FT-IR spectra of normal and glioma brain constituents such as lipids, DNA, and proteins (measured on delipidized tissue). Modeling of FT-IR spectra yielded fit coefficients reflecting the chemical changes associated with a tumor. Our results demonstrate the ability of FT-IRM to assess the importance and distribution of each individual constituent and its variation in normal brain structures as well as in the different pathological states of glioma. We demonstrated that (i) cholesterol and phosphatidylethanolamine contributions are highest in corpus callosum and anterior commissure but decrease gradually towards the cortex surface as well as in the tumor, (ii) phosphatidylcholine contribution is highest in the cortex and decreases in the tumor, (iii) galactocerebroside is localized only in white, but not in gray matter, and decreases in the vital tumor region while the necrosis area shows a higher concentration of this cerebroside, (iv) DNA and oleic acid increase in the tumor as compared to gray matter. This approach could, in the future, contribute to enhance diagnostic accuracy, improve the grading, prognosis, and play a vital role in therapeutic strategy and monitoring.

The advent of moderate dilatations in ascending aortas is often accompanied by structural modifications of the main components of the aortic tissue, elastin and collagen. In this study, we have undertaken an approach based on FTIR microscopy coupled to a curve-fitting procedure to analyze secondary structure modifications in these proteins in human normal and pathological aortic tissues. We found that the outcome of the aortic pathology is strongly influenced by these proteins, which are abundant in the media of the aortic wall, and that the advent of an aortic dilatation is generally accompanied by a decrease of parallel beta-sheet structures. Elastin, essentially composed of beta-sheet structures, seems to be directly related to these changes and therefore indicative of the elastic alteration of the aortic wall. Conventional microscopy and confocal fluorescence microscopy were used to compare FTIR microscopy results with the organization of the elastic fibers present in the tissues. This in-vitro study on 6 patients (three normal and three pathologic), suggests that such a spectroscopic marker, specific to aneurismal tissue characterization, could be important information for surgeons who face the dilemma of moderate aortic tissue dilatation of the ascending aortas.

Chloramphenicol (CLM), originally derived from the bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyl transferase activity. The near infrared Fourier transform (NIR-FT) Raman, surface-enhanced Raman spectroscopy (SERS) and Fourier transform infrared (FT-IR) spectral analyses of CLM, a potential antibacterial drug for the treatment of typhoid fever, were carried out along with density functional computations. The vibrational spectral analysis reveals that the CH2 asymmetric and symmetric stretching modes are shifted to higher wavenumbers than the computed values, owing to the electronic effects resulting from induction of methylene group with the adjacent electronegative atom. The lowering of CO stretching wavenumber is due to the presence of the strong electronegative atom, nitrogen, attached to the carbonyl carbon, causing large degree of molecular π-electron delocalization and redistribution of electrons, which weakens the CO bond. The absence of a CH stretching vibration and the observed CH out-of-plane bending modes suggest that the CLM molecule may be adsorbed in a flat orientation with respect to the silver surface. Copyright © 2008 John Wiley & Sons, Ltd.

Resume La spectroscopie Raman est une technique efficace d’analyse de la composition moleculaire d’echantillons biologiques. Des methodes de separation de sources peuvent etre utilisees pour separer efficacement les informations denses melangees dans les spectres Raman acquis. Des deformations telles que le fond de fluorescence, le desalignement des pics ou l’heterogeneite de la largeur des pics remettent en cause le modele generatif lineaire requis par les methodes de separation de sources. Des pretraitements sont necessaires pour compenser ces effets perturbateurs et ainsi rendre les spectres Raman descriptibles par un modele generatif lineaire. Nous montrons dans cet article que l’efficacite des methodes de separation de sources est profondement dependante des pretraitements appliques aux donnees. L’etude du deparaffinage numerique du signal Raman d’une biopsie de peau humaine prouve les ameliorations ainsi apportees. Les methodes de separation de sources appliquees dans cet article sont : un algorithme classique d’analyse en composantes independantes (ACI), nomme joint approximate diagonalization of eigenmatrices (JADE) et deux methodes de separation de sources positives, appelees la factorisation en matrices non-negatives (FMN) et la separation de sources positives par maximum de vraisemblance (SSPMV).