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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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...

FTIR spectral imaging was applied on formalin-fixed paraffin-embedded biopsies from colon and skin cancerous lesions. These samples were deposited onto different substrates (zinc selenide and calcium fluoride respectively) and embedded using two types of paraffin. Formalin fixation followed by paraffin embedding is the gold standard in tissue storage. It can preserve molecular structures and it is compatible with immunohistochemistry. However, paraffin absorption bands are significant in the mid-infrared region and can mask some molecular vibrations of the tissue. Direct data processing was applied on spectral images without any chemical dewaxing of the tissues. Extended Multiplicative Signal Correction was used to correct the spectral contribution from paraffin. For this purpose, the signal of paraffin was modelled using Principal Component Analysis and paraffin spectra were removed from the raw images based on an outlier detection. Then, pseudo-colour images were computed by K-means clustering in order to highlight histological structures of interest. This robust chemometrics methodology was applied on the two samples. Tumour areas were successfully demarcated from the rest of the tissue in both colon and skin independently of the embedding material and of the substrate.

The purpose of this study was to investigate molecular changes associated with glioma tissues by Raman microspectroscopy in order to develop its use in clinical practice. Spectroscopic markers obtained from C6 glioma tissues were compared to conventional histological and histochemical techniques. Cholesterol and phospholipid contents were highest in corpus callosum and decreased gradually towards the cortex surface as well as in the tumor. Two different necrotic areas have been identified: a fully necrotic zone characterized by the presence of plasma proteins and a peri-necrotic area with a high lipid content. This result was confirmed by Nile Red staining. Additionally, one structure was detected in the periphery of the tumor. Invisible with histopathological hematoxylin and eosin staining, it was revealed by immunohistochemical Ki-67 and MT1-MMP staining used to visualize the proliferative and invasive activities of glioma, respectively. Hierarchical cluster analysis on the only cluster averaged spectra showed a clear distinction between normal, tumoral, necrotic and edematous tissues. Raman microspectroscopy can discriminate between healthy and tumoral brain tissue and yield spectroscopic markers associated with the proliferative and invasive properties of glioblastoma. Development of in vivo Raman spectroscopy could thus accurately define tumor margins, identify tumor remnants, and help in the development of novel therapies for glioblastoma.

A non-optical force sensor that allows operation both in lateral (shear) and in vertical (tapping) force detection modes has been introduced for dynamic tip–sample distance regulation in scanning near-field optical microscopy (SNOM) of biological samples. The sensor is based on a rectangular bimorph cantilever consisting of two thin piezoceramic layers bonded to a brass centre shim. One of the piezo layers serves as the probe dither and another as the responder of the sensed forces. The sensor is driven with a home-made Q-control electronics so that its sensitivity and bandwidth can be adjusted. The dynamics, characteristics and design considerations of the sensor are theoretically and experimentally discussed. Driving the bimorph cantilever at its eigenfrequency with appropriate force feedback allows one to obtain a quality factor (Q-factor) up to 103 in water, suitable for different sample softness and imaging environments. The high sensitivity of the sensor is demonstrated both by shear force and by tapping mode imaging of soft biological samples in their natural state. Near-field optical resolution of better than 100 nm on red blood cells in water has been obtained. The experimental results suggest that this SNOM sensor would be a promising set-up for biological applications. Copyright © 2007 John Wiley & Sons, Ltd.

Optical histopathology is fast emerging as a potential tool in cancer diagnosis. Fresh tissues in saline are ideal samples for optical histopathology. However, evaluation of suitability of ex vivo handled tissues is necessitated because of severe constraints in sample procurement, handling, and other associated problems with fresh tissues. Among these methods, formalin-fixed samples are shown to be suitable for optical histopathology. However, it is necessary to further evaluate this method from the point of view discriminating tissues with minute biochemical variations. A pilot Raman and Fourier transform infrared (FTIR) microspectroscopic studies of formalin-fixed tissues normal, malignant, and after-2-fractions of radiotherapy from the same malignant cervix subjects were carried out, with an aim to explore the feasibility of discriminating these tissues, especially the tissues after-2-fractions of radiotherapy from other two groups. Raman and FTIR spectra exhibit large differences for normal and malignant tissues and subtle differences are seen between malignant and after-2-fractions of radiotherapy tissues. Spectral data were analyzed by principal component analysis (PCA) and it provided good discrimination of normal and malignant tissues. PCA of data of three tissues, normal, malignant, and 2-fractions after radiotherapy, gave two clusters corresponding to normal and malignant + after-2-fractions of radiotherapy tissues. A second step of PCA was required to achieve discrimination between malignant and after-2-fractions of radiotherapy tissues. Hence, this study not only further supports the use of formalin-fixed tissues in optical histopathology, especially from Raman spectroscopy point of view, it also indicates feasibility of discriminating tissues with minute biochemical differences such as malignant and after-2-fractions of radiotherapy.

Human skin is directly exposed to different exogenous agents. Many research works have studied the diffusion, interactions, absorption mechanisms, and/or toxicity of these agents toward different cutaneous structures. With the use of living animals for such tests being more and more rejected; and the number of human volunteers being limited; different types of skin models are used. In the last few years, reconstructed epidermis from cell cultures has been frequently employed, and recent changes in the European chemical policy have approved and encouraged the use of these reconstructed models for skin-related research works and assessments. Among the techniques used actually to study the skin, Raman microspectroscopy is a rising and powerful nondestructive technique that detects characteristic molecular vibrations. In this study, we created a spectral database to index the vibration peaks and bands of a well-known reconstructed epidermis model, the Episkin. The comparison with a native epidermis signal enabled us to put in evidence several spectral differences associated with molecular and structural differences between the skin and the reconstructed model, both maintained in living conditions. In addition to that, we have showed the feasibility of tracking the penetration of a pharmaceutical molecule through the Episkin model. (

// Marie Guilbert 1,* , Blandine Roig 2,* , Christine Terryn 3 , Roselyne Garnotel 1 , Pierre Jeannesson 1 , Ganesh D. Sockalingum 1,3 , Michel Manfait 1 , Francois Perraut 2 , Jean-Marc Dinten 2 , Anne Koenig 2 and Olivier Piot 1,3 1 MeDIAN-Biophotonique et Technologies pour la Sante, Universite de Reims Champagne-Ardenne, CNRS UMR 7369 MEDyC, UFR de Pharmacie, SFR CAP Sante, Reims, France 2 CEA, LETI, Minatec campus, Grenoble, France 3 Plate-forme Imagerie Cellulaire et Tissulaire, Universite de Reims Champagne-Ardenne, Reims, France * These authors have contributed equally to this work Correspondence to: Olivier Piot, email: // Keywords : Type I collagen aging, 3D matrix, confocal reflectance microscopy, diffuse reflectance spectroscopy, ATR infrared imaging, Gerotarget Received : September 07, 2015 Accepted : January 30, 2016 Published : February 14, 2016 Abstract During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

International audience; Upon chronological aging, human skin undergoes structural and molecular modifications, especially at the level of type I collagen. This macromolecule is one of the main dermal structural proteins and presents several age-related alterations. It exhibits a triple helical structure and assembles itself to form fibrils and fibers. In addition, water plays an important role in stabilizing the collagen triple helix by forming hydrogen-bonds between collagen residues. However, the influence of water on changes of dermal collagen fiber orientation with age has not been yet understood. Polarized-Fourier Transform Infrared (P-FTIR) imaging is an interesting biophotonic approach to determine in situ the orientation of type I collagen fibers, as we have recently shown by comparing skin samples of different ages. In this work, P-FTIR spectral imaging was performed on skin samples from two age groups (35-and 38-year-old on the one hand, 60-and 66-year-old on the other hand), and our analyses were focused on the effect of H 2 O/D 2 O substitution. Spectral data were processed with fuzzy C-means (FCM) clustering in order to distinguish different orientations of collagen fibers. We demonstrated that the orientation was altered with aging, and that D 2 O treatment, affecting primarily highly bound water molecules, is more marked for the youngest skin samples. Collagen-bound water-related spectral markers were also highlighted. Our results suggest a weakening of water/collagen interactions with age. This non-destructive and label-free methodology allows us to understand better the importance of bound water in collagen fiber orientation alterations occurring with skin aging. Obtaining such structural information could find benefits in dermatology as well as in cosmetics. † Electronic supplementary information (ESI) available. See

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.

Ovarian cancer is the sixth most common cancer among women worldwide, and mortality rates from this cancer are higher than for other gynecological cancers. This is attributed to a lack of reliable screening methods and the inadequacy of treatment modalities for the advanced stages of the disease. FTIR and Raman spectroscopic studies of formalin-fixed normal, benign, and malignant ovarian tissues have been undertaken in order to investigate and attempt to understand the underlying biochemical changes associated with the disease, and to explore the feasibility of discriminating between these different tissue types. Raman spectra of normal tissues indicate the dominance of proteins and lower contents of DNA and lipids compared to malignant tissues. Among the pathological tissues studied, spectra from benign tissues seem to contain more proteins and less DNA and lipids compared to malignant tissue spectra. FTIR studies corroborate these findings. FTIR and Raman spectra of both normal and benign tissues showed more similarities than those of malignant tissues. Cluster analysis of first-derivative Raman spectra in the 700-1700 cm(-1) range gave two clear groups, one corresponding to malignant and the other to normal+benign tissues. At a lower heterogeneity level, the normal+benign cluster gave three nonoverlapping subclusters, one corresponding to normal and two for benign tissues. Cluster analysis of second-derivative FTIR spectra in the combined spectral regions of 1540-1680 and 1720-1780 cm(-1) resulted into two clear clusters corresponding to malignant and normal+benign tissues. The cluster corresponding to normal+benign tissues produced nonoverlapping subclusters for normal and benign tissues at a lower heterogeneity level. The findings of this study demonstrate the feasibility of Raman and FTIR microspectroscopic discrimination of formalin-fixed normal, benign, and malignant ovarian tissues.

Fourier transform infrared (FT-IR) spectroscopy is a valuable technique for characterization of biological samples, providing a detailed fingerprint of the major chemical constituents. However, water vapor and CO2 in the beam path often cause interferences in the spectra, which can hamper the data analysis and interpretation of results. In this paper we present a new method for removal of the spectral contributions due to atmospheric water and CO2 from attenuated total reflection (ATR)-FT-IR spectra. In the IR spectrum, four separate wavenumber regions were defined, each containing an absorption band from either water vapor or CO2. From two calibration data sets, gas model spectra were estimated in each of the four spectral regions, and these model spectra were applied for correction of gas absorptions in two independent test sets (spectra of aqueous solutions and a yeast biofilm ( C. albicans) growing on an ATR crystal, respectively). The amounts of the atmospheric gases as expressed by the model spectra were estimated by regression, using second-derivative transformed spectra, and the estimated gas spectra could subsequently be subtracted from the sample spectra. For spectra of the growing yeast biofilm, the gas correction revealed otherwise hidden variations of relevance for modeling the growth dynamics. As the presented method improved the interpretation of the principle component analysis (PCA) models, it has proven to be a valuable tool for filtering atmospheric variation in ATR-FT-IR spectra.

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950-1,185 cm(-1)), protein (1,480-1,720 cm(-1)), and the fatty acids (2,840-3,000 cm(-1)) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as beta-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.

In this paper, a shear force scanning near-field fluorescence microscope combined with a confocal laser microspectrofluorometer is described. The shear force detection is realized based on a bimorph cantilever, which provides a very sensitive, reliable, and easy to use method to control the probe-sample distance during scanning. With the system, high-quality shear force imaging of various samples has been carried out. Furthermore, simultaneous shear force and near-field fluorescence imaging of biological cells has also been realized. As an example, we especially present the result on the distribution of P-glycoprotein in the plasma membrane of human small cell lung cancer cells, suggesting that the system would be a promising tool for biological applications.

Lung cancer is a disease associated with a poor prognosis. One of the reasons for this is the lack of effective treatment. Chemotherapy, together with surgery and radiotherapy, is one of the main treatments for this disease. However, it is not possible at present to determine which tumours would be more sensitive to a given drug or combination of drugs. In this study, FT-IR microspectroscopy was used to assess lung cancer cells response to the chemotherapeutic agent gemcitabine. Studies on two lung cancer cell lines showed that treatment of in vitro growing lung cancer cells with gemcitabine caused changes in their FT-IR spectral pattern in the 950–1150 cm−1 region. An increase in the 1080 cm−1 peak was observed and the 1080 cm−1/1050 cm−1 ratio, corresponding to symmetric vibrations of PO2 groups, and C O stretches of carbohydrates moieties and glycogen, respectively, was found to increase with the dose of gemcitabine added. Furthermore, the 1080 cm−1/1050 cm−1 ratio reached a plateau phase when the lethal dose 75 (LD75) was reached in the case of A549 lung cancer cell line and between the LD50 and LD75 in the case of CALU-1 lung cancer cell line. The data here presented shows that it is possible not only to detect changes in the spectra of in vitro growing cancer cells following the addition of gemcitabine but also, to correlate these changes with cell survival.

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.

Lung cancer is usually fatal once it becomes metastatic. However, in order to develop metastases, a tumor usually invades the basal membrane and enters the vascular or lymphatic system. In this study, a three-dimensional artificial membrane using collagen type I, one of the main components of basal membranes, was established in order to investigate tumor cell invasion. Lung cancer cell line CALU-1 was seeded on this artificial membrane and cell invasion was studied using the Fourier transform infrared (FTIR) imaging technique. This approach allowed identification of tumor cells invading the collagen type I membrane by means of their infrared spectra and images. The mapping images obtained with FTIR microspectroscopy were validated with standard histological section analysis. The FTIR image produced using a single wavenumber at 1080 cm(-1), corresponding to PO2- groups in DNA from cells, correlated well with the histological section, which clearly revealed a cell layer and invading cells within the membrane. Furthermore, the peaks corresponding to amide A, I, and II in the spectra of the invading cells shifted compared to the noninvading cells, which may relate to the changes in conformation and/or heterogeneity in the phenotype of the cells. The data presented in this study demonstrate that FTIR microspectroscopy can be a fast and reliable technique to assess tumor invasion in vitro.

Micro-Raman spectra of formalin-fixed oral squamous normal and carcinoma tissues, stored at room temperature for 2 months, have been recorded. Spectra were recorded both in the epithelial and subepithelial regions of the tissues. No noticeable spectral contamination due to formalin was observed. Very significant differences between spectra of normal epithelial and malignant epithelial samples were found. No such differences in spectra of subepithelial malignant and subepithelial normal samples could be observed. This study shows that spectra from the epithelial region changes drastically because of malignancy-induced biochemical changes in this region. Major differences between normal and malignant spectra seem to arise from the protein composition, conformational/structural changes, and possible increase in protein content in malignant epithelia. The differences between normal epithelial and subepithelial spectra, as expected, arise mainly from the collagen in subepithelial tissue. Principal component analysis of the combined sets of spectra—epithelial and subepithelial, normal and malignant— showed that very good discrimination can be achieved by Raman microspectroscopy. This study thus validates the suitability of formalin-fixed tissues for optical pathology in oral malignancy.

Homocamptothecin (hCPT) is a new camptothecin (CPT) derivative with a seven-membered β-hydroxylactone E-ring. This modification provides higher lactone stability and did not impair its activity against topoisomerase I (top1), but rather appears to improve it compared to CPT. Such lactone modification was unexpected regarding the previous structure-activity relationship data inside the CPT series, and may have crucial mechanistic implications in the ternary cleavable complex formation. In this study, the detailed characterization of the E-ring homologation and lactone/carboxylate conversion, self-aggregation, influence of pH and polarity of the molecular environment have been performed for hCPT by frequency-domain fluorescence. The real-time spectrofluorometry confirmed the enhanced stability of hCPT. We have also investigated the E-ring status of hCPT within the top1 ternary complex with DNA, and with top1 or DNA binary complexes. Unlike CPT, no modification of the (β-hydroxy-) lactone-carboxylate conversion rates was observed, suggesting that E-ring opening is not required for cleavable complex stabilization in presence of hCPT. Comparison of the two structures by molecular modeling revealed similar conformation and steric volumes between the β-hydroxylactone ring conformation of hCPT and the opened ring of CPT. The lack of hCPT E-ring opening was discussed in the light of these molecular modeling results.

The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8+/-0.3 to 1.6+/-0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 microM thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.

International audience; During chronological skin aging, alterations in dermal structural proteins cause morphological modifications. Modifications are probably due to collagen fiber (type I collagen) rearrangement and reorientation with aging that have not been researched until now. FTIR microspectroscopy appears as an interesting method to study protein structure under normal and pathological conditions. Associated with a polarizer, this vibrational technique permits us to probe collagen orientation within skin tissue sections, by computing the ratio of integrated intensities of amide I and amide II bands. In this study, we used the polarized-FTIR imaging to evaluate molecular modifications of dermal collagen during chronological aging. The data processing of polarized infrared data revealed that type I collagen fibers become parallel to the skin surface in aged skin dermis. Our approach could find innovative applications in dermatology as well as in cosmetics.

Confocal Raman microspectroscopy has previously been employed to investigate the protein content and composition of the starchy endosperm of the wheat grain. With the same objective, that is to determine the molecular basis of grain cohesion and more specifically of kernel hardness, the contribution of endosperm cell walls in the kernel structure and cohesion was explored. The technique showed that endosperm cell walls consist not only of arabinoxylan chains with ramifications of ferulic esters, but also of others components such as proteins and lipids that could play some role in the mechanical properties of the endosperm cell walls. A new model of interaction between ferulic ramifications and a phospholipid component was proposed. The investigation of cell wall composition at successive stages of grain development revealed a decrease in the protein to arabinoxylan ratio and simultaneously an increase of the ferulic acid to arabinoxylan ratio that could be associated with a strengthening of the cell wall structure. The study confirms the effectiveness of confocal Raman microspectroscopy to approach the structure of wheat grain at the micrometer scale and to identify specific molecular factors responsible for grain cohesion and involved in the fracture modes generated during the milling process.

Multidrug-resistant cells are believed to contain a plasma-membrane-efflux pump which is hypothesized to expel anticancer drugs from the cytosol to the cell exterior. Many of these drugs are classified as weak bases whose binding to intracellular targets is pH-dependent. Slight alterations in intracellular pH gradients have been shown to affect accumulation, endocytosis and secretion of drugs. In this study, we developed a new method based on confocal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine conjugated to dextran (FRD) and SNAFL-calcein-AM were used to determine pH in acidic compartments. Carboxy-SNARF1-AM was used to examine cytosolic pH. We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that corresponding to drug-resistant derivatives. Moreover, results obtained with carboxy-SNARF1-AM show that resistant cells display a more alkaline cytosolic pH. This results in a considerably larger pH gradient between the vesicular compartments and the cytosol of resistant cells than of sensitive cells. The lower pH gradient observed in sensitive cells may be related to a disruption in the organization of the trans-Golgi network (TGN). In drug-resistant cells, the organization of TGN appears compact. In addition, confocal microscopic analysis of cells labelled with FRD and SNAFL-calcein showed that sensitive cells contain a lower number of acidified vesicles. This suggest a diminished capacity of these cells to remove protonated drugs from the cytoplasm to secretory compartments followed by their secretion through the activity of the secretory and recycling pathways.

Fluorescence correlation spectroscopy (FCS) has been used to analyze the plasma membrane fluidity and heterogene- ity of multidrug resistant cells. At the single cell level, the effects of different membrane agents present in the extra-cellular medium have been explored. Firstly, we reveal a modification of plasma membrane heterogeneities according to the addition of a fluidity modulator, benzyl alcohol. On the other hand, revertants such as verapamil and cyclosporin-A appear to act more specifically on the slow diffusion sites, such as lipids microdomains.

We describe the effect strongly influencing the biological activity of some camptothecin (CPT) drugs, the inhibitors of DNA topoisomerase I (topo I), namely, the formation of J-type aggregates in an aqueous buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20-S-camptothecin (20(S)CPT), 10,11-methylenedioxy-CPT (10,11-CPT) and 7-ethyl-10-hydroxy-CPT (SN38). The aggregates were found to be stereospecific, not being detectable for the 20(R)-stereoisomer of CPT. They were formed by the stacking interaction between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. The aggregates were stable at acidic and neutral pHs, but dissociated at basic pHs. Self-aggregation prevented hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. Addition of BSA did not induce either disaggregation or hydrolysis of the lactone ring, whereas the monomeric form of the drugs was shown to undergo rapid conversion to an inactive carboxylate form in the presence of human serum albumin [5]. The drugs did not form the aggregates in the presence of topo I. Moreover, rapid dissociation of the aggregates was observed if a self-aggregated drug solution was added to topo I alone or to the DNA-topo I cleavage assay. Neither DNA alone nor oligonucleotides derived from the sequences of the CPT-enhanced or topo I-induced cleavage sites in SV40 plasmid DNA induces changes in the aggregation state of the drugs. These observations are indicative of interaction between the aggregates and topo I. The aggregates were found to penetrate within the cells with much higher efficiency than a monomeric form of the drugs. Cellular uptake of aggregated and nonaggregated species correlated well with cytotoxic effects produced by the drug. In this manner, CPT's self-aggregation should be regarded as a favourable phenomenon producing species with a more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.

Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.

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

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

Hyphenation of attenuated total reflection Fourier transform infrared spectroscopy and cluster analysis has been used to characterise a susceptible Escherichia coli K12 strain and the transconjugants TEM-1, TEM-2, TEM-3, SHV-2, SHV-3, SHV-4. A good discrimination of the susceptible strain from the transconjugants was obtained. Although a limited success was achieved in the differentiation of SHV and TEM phenotypes in general, results obtained with TEM-2 and SHV-3 were convincing. Spectral differences observed are ascribed to the global effects of the conjugation process, particularly their repercussions in the nucleic acids and carbohydrate absorbing regions, rather than to β-lactamase point-mutations.