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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.

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.

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.

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.

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.

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 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.

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.

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.

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.

Within the framework of the detection of toxic substances using specific bacteria, the effect of the toxic chemical compound tributyltin chloride (TBT) on the bacterium Escherichia coli TBT3 was investigated using a noninvasive approach based on micro‐Raman spectroscopy. Various cellular densities with optical density (OD) ranging from 1 to 4 and harvested at different growth phases were induced for 1 h with different TBT concentrations: 1, 3, 5, and 10 µM. As an original contribution, the Raman results show that, in these conditions, the vibrational signatures of bacteria were affected by the presence of the toxic TBT compound, mainly in the signal characteristics from carbohydrates and lipids. Hierarchical cluster analysis (HCA) carried out on the Raman data in these regions makes it possible to classify the spectra in three groups according to the TBT concentration, confirming the progressive influence of the chemical pollutant on the bacterium. This work shows the potentiality for the Raman t...

Raman spectra provide wealthy but complex information about the chemical constituents of biological samples. Digital processing techniques are usually needed to extract the spectra of chemical constituents and their associated concentration profiles. However, spectral signatures may admit transformations from those recorded on pure constituents and these techniques require a priori knowledge of spectra to be estimated. We propose in this study to analyse paraffin-embedded skin biopsies of malignant and benign tumors dedicated to oncology researches by Raman spectroscopy and advanced signal processing methods. We show that the commonly used principal component analysis (PCA) does not give physically interpretable estimators of spectra and associated concentration profiles. Based on a linear model and taking into account the statistical properties of spectra, independent component analysis (ICA) is used to better estimate the spectra of chemical constituents. The estimators of associated concentration profiles are no longer orthogonal and have only positive values, contrary to PCA. ICA allows to model the paraffin by three Raman spectra and provides good estimators of underlying spectra of the human skin, which is of great interest in oncology since the retrieval of spectral features of different types of skin tumors is sufficient for their discrimination.

Light/tissue interactions, like diffuse reflectance, endogenous fluorescence and Raman scattering, are a powerful means for providing skin diagnosis. Instrument calibration is an important step. We thus developed multilayered phantoms for calibration of optical systems. These phantoms mimic the optical properties of biological tissues such as skin. Our final objective is to better understand light/tissue interactions especially in the case of confocal Raman spectroscopy. The phantom preparation procedure is described, including the employed method to obtain a stratified object. PDMS was chosen as the bulk material. TiO2 was used as light scattering agent. Dye and ink were adopted to mimic, respectively, oxy-hemoglobin and melanin absorption spectra. By varying the amount of the incorporated components, we created a material with tunable optical properties. Monolayer and multilayered phantoms were designed to allow several characterization methods. Among them, we can name: X-ray tomography for structural information; Diffuse Reflectance Spectroscopy (DRS) with a homemade fibered bundle system for optical characterization; and Raman depth profiling with a commercial confocal Raman microscope for structural information and for our final objective. For each technique, the obtained results are presented and correlated when possible. A few words are said on our final objective. Raman depth profiles of the multilayered phantoms are distorted by elastic scattering. The signal attenuation through each single layer is directly dependent on its own scattering property. Therefore, determining the optical properties, obtained here with DRS, is crucial to properly correct Raman depth profiles. Thus, it would be permitted to consider quantitative studies on skin for drug permeation follow-up or hydration assessment, for instance.

Confocal Raman microspectroscopy allows in-depth molecular and conformational characterization of biological tissues non-invasively. Unfortunately, spectral distortions occur due to elastic scattering. Our objective is to correct the attenuation of in-depth Raman peaks intensity by considering this phenomenon, enabling thus quantitative diagnosis. In this purpose, we developed PDMS phantoms mimicking skin optical properties used as tools for instrument calibration and data processing method validation. An optical system based on a fibers bundle has been previously developed for in vivo skin characterization with Diffuse Reflectance Spectroscopy (DRS). Used on our phantoms, this technique allows checking their optical properties: the targeted ones were retrieved. Raman microspectroscopy was performed using a commercial confocal microscope. Depth profiles were constructed from integrated intensity of some specific PDMS Raman vibrations. Acquired on monolayer phantoms, they display a decline which is increasing with the scattering coefficient. Furthermore, when acquiring Raman spectra on multilayered phantoms, the signal attenuation through each single layer is directly dependent on its own scattering property. Therefore, determining the optical properties of any biological sample, obtained with DRS for example, is crucial to correct properly Raman depth profiles. A model, inspired from S.L. Jacques's expression for Confocal Reflectance Microscopy and modified at some points, is proposed and tested to fit the depth profiles obtained on the phantoms as function of the reduced scattering coefficient. Consequently, once the optical properties of a biological sample are known, the intensity of deep Raman spectra distorted by elastic scattering can be corrected with our reliable model, permitting thus to consider quantitative studies for purposes of characterization or diagnosis.

Background: Topical delivery of molecules into the human skin is one of the main issues in dermatology and cosmetology. Several techniques were developed to study molecules penetration into the human skin. Although widely accepted, the conventional methods such as Franz diffusion cells are unable to provide the accurate localization of actives in the skin layers. A different approach based on Raman spectroscopy has been proposed to follow-up the permeation of actives. It presents a high molecular specificity to distinguish exogenous molecules from skin constituents. Methods: Raman micro-imaging was applied to monitor the skin penetration of hyaluronic acids (HA) of different molecular weights. The first step, was the spectral characterization of these HA. After, we have determined spectral features of HA by which they can be detected in the skin. In the second part, transverse skin sections were realized and spectral images were recorded. Results: Our results show a difference of skin permeation of the three HA. Indeed, HA with low molecular weight (20– 300 kDa) passes through the stratum corneum in contrast of the impermeability of high molecular weight HA (1000– 1400 kDa). Conclusion: Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.

Raman spectroscopy is employed to record spectra which highlight vibrational information of biological structures. These spectra provide useful information about molecular composition of a tissue. However, the tissues are usually embedded into paraffin for a preservation purpose. The Independent Component Analysis (ICA) technique can be used to numerically dewax Raman spectra and to extract the specific information of the tissue. We show in this paper that the paraffin must be modeled by a three-source model in order to succeed. This linear model, confirmed also by analyzing paraffin blocks exclusively, should be preferred instead of commonly used one-source model.

Micro-Raman spectroscopy was used to investigate randomly mixed cell populations of human promyelocytic leukemia (HL60) and human breast cancer (MCF7); human uterine sarcoma (Mes-sa) and MCF7; as well as their respective pure cell lines. In this study the efficiency of micro-Raman spectroscopy to identify a cell type in randomly distributed mixed cell population was assessed. Raman data show that the differences in spectral profile between MCF7 and HL60 cell lines were more marked than those between MCF7 and Mes-sa cells. This shows that cells from different origins can display variances in their spectral signatures. Spectra were also analyzed by principal components analysis and results obtained from pure cell populations gave a reasonably good delineation between the cell types. Analysis of both mixed cell populations along with their pure cells counterparts, resulted in each case in three different clusters corresponding to the two pure cell populations and the mixed populations. However, a few spectra from the mixed population remained misclassified and were found to be closer to the clusters corresponding to pure cells. These results indicate that micro-Raman spectroscopy can be used to identify a cell type in a mixed cell population via its spectral signature.

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the β-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 297–302, 1998

Lactone and carboxylate forms of potent antitumor agents, camptothecins (CPTs) have been studied by Raman, Fourier-transform Raman (FT-Raman) and surface-enhanced Raman scattering (SERS) spectroscopy. Similarity of the Raman spectra of CPTs with corresponding FT-Raman spectra in the near-infrared allowed the latter to be compared with their SERS counterparts in order to analyse the interaction of the drugs with silver colloids. Different types of silver colloids (reduced with sodium borohydride or sodium citrate, with or without activation by anions) have been evaluated. Citrate-reduced colloid, activated with chloride anions (CAS) has been found to be the best compromise for SERS studies of both forms of CPTs. We suggest that in general CPTs are adsorbed on CAS via the nitrogen in ring B and are more inclined to a flat orientation than to a perpendicular one. However, probable interactions of substitution groups and/or of the COO− groups of hydrolysed CPTs with the CAS surface introduce some particularities in the adsorption patterns. As a result, SERS spectra are highly sensitive to hydrolysis and substitutions at distant rings of CPT and uniquely characteristic of each of the CPT derivatives. The pronounced hydrolysis-induced changes, similar in the Raman and SERS spectra of CPTs, involve similar vibrations in the spectra of different CPTs. Vibrational assignments, proposed for the main Raman and SERS bands of CPT and its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38 and topotecan) indicate that most of the bands which decreased upon lactone hydrolysis are those preferentially related to stretching modes of the quinoline rings A and B, and the bands which increased are those of the ring D stretching modes. Our data make the spectroscopic approach very promising for the further investigations of the molecular mechanisms of biological activity of CPTs.

Fourier transform (FT) Raman and surface enhanced Raman microspectroscopy (SERS) were used as a vibrational probe for investigating, in vitro and at the cellular level, the molecular interaction of dimethylcrocetin (DMCRT) with the retinoic acid nuclear receptor RAR-γ. FT-SERS results obtained in vitro with silver colloids demonstrated that DMCRT interacts specifically with RAR-γ. Comparison between the spectra of DMCRT in HL60 cancer cells and the in vitro DMCRT–RARγ complex showed practically the same shift in wavenumber of the band at 1210 cm-1 and the same intensity ratios I1541/I1165 and I1541/I1210. The similarity between the signal of DMCRT in K562 cells and in the free form is explained by either an absence of nuclear receptor or an absence of any interaction between the drug and receptor. These results seem to indicate that carotenoids could possibly activate the nuclear receptor in a similar manner to retinoids. © 1997 by John Wiley & Sons, Ltd.

Red and near-infrared excited Fourier transform surface-enhanced Raman spectra of an anticancer agent, all-trans retinoic acid (ATRA), adsorbed on gold island films are reported. Best results have been obtained with plates 80 A and 40 A thick respectively in the red and near-infrared and at concentrations of 10−5 and 5 × 10−6 M with a spinning system. The use of near-infrared laser excitation with low photon energy, allows us to overcome the problems of isomerisation when the sample is exposed for a long time to the laser radiation. Comparison between the Raman and SERS spectra in the visible shows that the adsorption on the surface does not perturb the structure of ATRA and confirms the long range enhancement of the island films with this type of molecule. Spectral data show that while gold island films and colloids are appropriate substrates for use with red excitation, silver and gold colloids as well as gold island films exhibit satisfactory enhancement levels in the near-infrared. This study will in the future allow us to choose the appropriate system that will serve to investigate the interaction of ATRA with its target in vitro and the effect of this differentiating agent in human leukaemia cell lines such as K562 and HL60.

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.

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.

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.

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.

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.

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.

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).

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.

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 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.

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.

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

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.

Raman spectra are classically modeled as a linear mixing of spectra of molecular constituents of the analyzed sample. Source separation methods are thus well suited to estimate these constituent spectra. However, physical distortions due to the instrumentation and biological nature of samples add nonlinearities to the Raman spectra model. These distortions are dark current, detector and optic responses, fluorescence background, and peak misalignment and peak width heterogeneity. The source separation results are thus deteriorated by these effects. We propose to develop specific preprocessing steps to correct these distortions and to retrieve a linear model. The benefits brought by these steps are studied by the application of two different source separation methods named joint approximate diagonalization of eigenmatrices and maximum likelihood positive source separation after the application of each step on a dataset acquired on a paraffin-embedded human skin biopsy. The efficacy of these methods to separate Raman spectra is also discussed.