Conformation changes in human hair keratin observed using confocal Raman spectroscopy after active ingredient application.

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 ß-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 ß-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 ß-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.

OBJECTIF: Dans l'évaluation des produits cosmétiques pour le soin des cheveux, une méthode communément utilisée consiste à évaluer l'aspect des cheveux afin de déterminer l'effet d'un principe actif sur l'état final des cheveux via l'appréciation visuelle. Bien que d'autres techniques ont été proposées pour une analyse directe de la fibre capillaire, elles ne donnent que des informations de surface ou de structure, sans aucune information moléculaire précise. Une approche différente par la spectroscopie confocale Raman a été proposée pour le suivi in situ du changement moléculaire de la kératine directement dans les fibres de cheveux humains. Il présente une grande spécificité moléculaire, détecter les interactions chimiques entre les molécules et peut fournir des informations moléculaires à différents niveaux de profondeur du cortex et de la cuticule. MÉTHODES: Afin d'évaluer le potentiel de la spectroscopie Raman confocale pour tester l'efficacité des ingrédients cosmétiques sur la structure de la kératine, nous avons entrepris une étude pilote sur l'efficacité d'un shampooing lissant sur cheveux naturels, en analysant les marqueurs spectraux de l'hélice α et du feuillet ß dans la bande Amide I et les marqueurs spectraux spécifiques au contenu en sulfure-cystine. RÉSULTATS: Nous avons confirmé qu'un actif s'avérant efficace sur un gold standard diminue la conformation de la kératine en hélice α et favorise la conformation de la kératine en feuillet ß dans les fibres des cheveux. Nous avons également montré que le traitement avec l'actif efficace diminue l'intensité des bandes de cystéine réticulant sous forme de ponts disulfures covalents (S - S à 510 cm-1). Ces données confirment que l'actif efficace agit également sur la structure tertiaire de la kératine. CONCLUSION: À partir de ces expériences, nous avons conclu que l'actif efficace a un effet lissant sur les fibres du cheveu humain en agissant sur la conformation hélice α et feuillet ß de la kératine et sur la structure tertiaire de la kératine. Sur la base de ces résultats, la spectroscopie confocale Raman peut être considérée comme une technique puissante pour étudier l'influence des ingrédients cosmétiques sur la structure de la kératine dans les fibres de cheveux. De plus, cette technique analytique a l'avantage d'être non destructive et ne nécessite pas de marquage; de plus, elle ne nécessite pas d'extraction ou de purification des échantillons et peut être appliquée en routine dans les laboratoires de cosmétiques.

Lipid organization in xerosis: the key of the problem?

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.

Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy.

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.

Implementation of an FTIR spectral library of 486 filamentous fungi strains for rapid identification of molds.

Filamentous fungi may cause food and feed spoilage and produce harmful metabolites to human and animal health such as mycotoxins. Identification of fungi using conventional phenotypic methods is time-consuming and molecular methods are still quite expensive and require specific laboratory skills. In the last two decades, it has been shown that Fourier transform infrared (FTIR) spectroscopy was an efficient tool for microorganism identification. The aims of this study were to use a simple protocol for the identification of filamentous fungi using FTIR spectroscopy coupled with a partial least squares discriminant analysis (PLS-DA), to implement a procedure to validate the obtained results, and to assess the transferability of the method and database. FTIR spectra of 486 strains (43 genera and 140 species) were recorded. An IR spectral database built with 288 strains was used to identify 105 different strains. It was found that 99.17% and 92.3% of spectra derived from these strains were correctly assigned at the genus and species levels, respectively. The establishment of a score and a threshold permitted to validate 80.79% of the results obtained. A standardization function (SF) was also implemented and tested on FTIR data from another instrument on a different site and permitted to increase the percentage of well predicted spectra for this set from 72.15% to 89.13%. This study confirms the good performance of high throughput FTIR spectroscopy for fungal identification using a spectral library of molds of industrial relevance.

Vibrational spectroscopy coupled to classical least square analysis, a new approach for determination of skin moisturizing agents' mechanisms.

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.

Diagnosis of hepatocellular carcinoma in cirrhotic patients: a proof-of-concept study using serum micro-Raman spectroscopy.

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

Histopathological characterization of primary cutaneous melanoma using infrared microimaging: a proof-of-concept study.

BACKGROUND: The diagnosis of malignant melanoma is based upon the histological evaluation of the lesion. As such, the morphological interpretation relies on the expertise of a dermatopathologist. Infrared microimaging is emerging as a new powerful tool to investigate tissue biochemistry. Infrared spectra probe the biochemical constitution of the sample and are real tissue-specific spectroscopic fingerprints. OBJECTIVES: To assess the potential of infrared microimaging to aid in the analysis of tissue sections from primary cutaneous melanomas. METHODS: Ten samples of melanoma sections from the main histological subtypes were investigated using infrared microimaging combined with multivariate statistical analyses. RESULTS: This methodology yielded highly contrasted colour-coded images that permitted to highlight tissue architecture without any staining. It was possible to discriminate tumour areas from normal epidermis automatically, and intratumoral heterogeneity as revealed by our approach was correlated with the aggressiveness of the tumour. CONCLUSIONS: This proof-of-concept study shows that infrared microimaging could help in the diagnosis of primary cutaneous melanoma.

Activated sludge behaviour in a batch reactor in the presence of antibiotics: study of extracellular polymeric substances.

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.

Detection of pathological aortic tissues by infrared multispectral imaging and chemometrics.

Processing of multispectral images is becoming an important issue, especially in terms of data mining for disease diagnosis. We report here an original image analysis procedure developed in order to compare 42 infrared multispectral images acquired on human ascending aortic healthy and pathological tissues. Each image contained about 2500 infrared absorption spectra, each composed of 1641 variables (wavenumbers). To process this large data set, we have restricted the spectral window used to the 1800-950 cm(-1) spectral range and selected 100 spectra from the aortic media, which is the most altered part of the aortic tissue in aneurysms. Prior to this selection, a spectral quality test was performed to eliminate 'bad' spectra. Our data set was first subjected to a discriminant analysis, which allowed separation of aortic tissues in two groups corresponding respectively to normal and aneurysmal states. Then a K-means analysis, based on 20 groups, allowed reconstruction of infrared images using false-colours and discriminated between pathological and healthy tissues. These results demonstrate the usefulness of such data processing methods for the analysis and comparison of a set of spectral images.

In-vitro analysis of normal and aneurismal human ascending aortic tissues using FT-IR microspectroscopy.

FTIR microspectroscopy has shown to be a proven tool in the investigation of many tissue types. We have used this spectroscopic approach to analyse structural differences between normal and aneurismal aortic tissues and also aortas from patients with congenital anomalies like aortic bicuspid valves. Spectral analysis showed important variations in amide I and II regions, related to changes in alpha-helix and beta-sheet secondary structure of proteins that seem to be correlated to structural modifications of collagen and elastin. These proteins are the major constituents of the aortic wall associated to smooth muscular cells. The amide regions have thus been identified as a marker of structural modifications related to these proteins whose modifications can be associated to a given aortic pathological situation. Both univariate (total absorbance image and band ratio) and multivariate (principal components analysis) analyses of the spectral information contained in the infrared images have been performed. Differences between tissues have been identified by these two approaches and allowed to separate each group of aortic tissues. However, with univariate band ratio analysis, the pathological group was found to be composed of samples from aneurismal aortas associated or not with an aortic bicuspid valve. In contrast, PCA was able to separate these two types of aortic pathologies. For other groups, PCA and band ratio analysis can differentiate between normal, aneurismal, and none dilated aortas from patients with a bicuspid aortic valve.

Characterisation of uterine sarcoma cell lines exhibiting MDR phenotype by vibrational spectroscopy.

Multidrug resistance (MDR) enables cancer cells to escape cytotoxic insults of anticancer drugs. Rapid identification of cells exhibiting the MDR phenotype is very important since it can lead to an effective and individual patient based treatment plan. We have investigated a combined vibrational spectroscopic approach, using both micro-Raman and FTIR techniques, in order to characterise a sensitive human uterine sarcoma cell line MES-SA and its multidrug-resistant derivative Garf. In this study, these two complementary methods have been evaluated via the use of principal components analysis (PCA), for discrimination of cells exhibiting the MDR phenotype. Our results indicate that, though they inherently have different sensitivities, both Raman and IR methods can provide a good differentiation of cell phenotypes.

Ultrastructural appraisal of the multidrug resistance in K562 and LR73 cell lines from Fourier transform infrared spectroscopy.

Two different cell lines sharing the multidrug resistance (MDR) phenotype were investigated for 8 months by means of Fourier transform IR spectroscopy on cell smears. We studied (a) a human leukemic doxorubicin-sensitive K562 cell line, from which a doxorubicin-resistant K562 cell subline was subsequently derived; (b) a Chinese hamster LR73 drug-sensitive line, subsequently transfected with the expression plasmid pDREX4 containing the mdr1 gene, to produce a multidrug-resistant LR73 subline (MDR-LR73). The sensitivity of Fourier transform IR spectroscopy has allowed differentiation between sensitive and MDR phenotypes among the above lines, even in double blind studies. The MDR phenotype is characterized by three combined features in spectra: (a) a decrease in the intensities of the amide I and II bands; (b) a shoulder on the high wave numbers slope of the amide I bands; (c) a shift toward the high wave numbers of the amide II bands. Furthermore, computational treatment of Fourier transform IR spectra (deconvolution and Gaussian curve-fitting techniques), has evidenced, in MDR-K562 and MDR-LR73 cell sublines, a conformational change involving the same protein in both sublines. It is hypothesized that the protein implicated in the conformational change may be related to the MDR phenotype.

Molecular and cellular interactions between intoplicine, DNA, and topoisomerase II studied by surface-enhanced Raman scattering spectroscopy.

The surface-enhanced Raman scattering spectra of the new antitumoral agent, intoplicine (RP 60475, NSC 645008), and those of its complexes with DNA and topoisomerase II in vitro and in K562 cancer cells were obtained. Intoplicine was found to unwind DNA and to inhibit purified calf thymus topoisomerase II via a stabilization of the ternary cleavable complex. The intensity of the surface-enhanced Raman scattering spectrum of intoplicine was not modified by the addition of plasmid pBR322 or calf thymus DNA. In the complex of this antitumor agent with topoisomerase II, the signal of intoplicine was completely abolished, indicating that at least some portion of intoplicine binds to an internal part of the enzyme. During the formation of the ternary complex, intoplicine was released from the interior of the protein and formed hydrogen bonds via its hydroxyl and/or amino groups. Similar modifications of the intoplicine spectra were found by microsurface-enhanced Raman scattering spectroscopy of the compound in the nucleus of treated K562 cells. In contrast, intoplicine was found to be in a free form in the cytoplasm.

Mechanisms of resistance of confluent human and rat colon cancer cells to anthracyclines: alteration of drug passive diffusion.

Two colon cancer cell lines, HT-29 (human) and DHD/K12/TRb (rat), were grown as monolayer cultures to various confluence degrees. The cytotoxic efficacies of doxorubicin and 4'-deoxydoxorubicin, evaluated by a survival assay, and the nuclear drug concentrations, measured by microspectrofluorometry, were shown to progressively decrease with the augmentation of confluence. This confluence dependent resistance (CDR) to anthracyclines was demonstrated independent of the multidrug resistance drug efflux mechanism. The cellular uptake of three compounds (sodium [51Cr]chromate, D-[14C]alanine, L-[14C]glucose) known to passively diffuse across the cell membrane as anthracyclines do was also reduced in confluent cells. After trypsin cell detachment, the kinetics of reversion of the sodium [51Cr]chromate uptake decrease and that of CDR were similar. Therefore, CDR may be attributed to a reduction of anthracycline cell intake due to a general alteration of passive diffusion across the cell membrane. However, CDR is only partly explained by this phenomenon since a reduced sensitivity of confluent cells was observed compared with nonconfluent cells for a similar amount of drug in their nuclei. CDR could explain the high resistance to anthracyclines of some solid tumors, such as colon tumors, in which cancer cells are tightly aggregated.

Correlation between growth inhibition and intranuclear doxorubicin and 4'-deoxy-4'-iododoxorubicin quantitated in living K562 cells by microspectrofluorometry.

Intranuclear drug concentration in cells treated with doxorubicin (DXR) or with 4'-deoxy-4'-iododoxorubicin (IDX) was measured by means of a quantitative microspectrofluorometric technique recently developed by us. Resolution of free and bound drug contributions in fluorescence emission spectra, as collected from a microvolume of single living cell nuclei, provided concentration data with about 10% indetermination. Uptake of DXR and IDX into the nucleus of K562 cells and DXR-resistant K562/DXR cells could then be studied with a sensitive, nondestructive technique. Growth inhibitory concentrations of K562 and K562/DXR cells, when measured with respect to drug content in the medium, differed by a factor of 25 in the case of DXR and by a factor of three in the case of IDX. By contrast, intranuclear drug concentrations measured at corresponding growth inhibitory concentrations are found to be nearly constant, i.e., independent of cellular-resistant phenotype and of anthracycline structure. This result supports an identical mechanism of action for the two drugs, most probably targeted to the nucleus, and ascribes to intracellular transport the different potency of the two drugs in the two cell lines.

[In vivo functional microspectroscopy of cells and tissues]. / Microspectroscopies fonctionnelles du vivant. Approches cellulaires et tissulaires.

The interaction between living matter and optical spectra (Raman, infrared, fluorescence spectroscopy) can be used to characterize simple or complex biomolecular systems. At the cell and tissue level, these microspectroscopic techniques can be used to construct descriptors of molecular or supramolecular (nucleic acids, proteins, lipids, etc.) function in a given biological situation (healthy or pathological tissue, normal or pathological cell, induction of cytotoxicity, apoptosis or differentiation, presence or not of a resistance phenotype, etc.) At the present time vibrational spectroscopy appears to be a particularly powerful technique for characterization of the early phases of pathological processes occurring within given cells or tissues. It could be used to develop tools for early diagnosis and prediction of therapeutic response.

Conformational changes of human serum albumin in vivo induced by free fatty acids as studied by Fourier transform infrared spectroscopy.

Binding of free fatty acids (FFA) to human serum albumin (HSA) was studied by Fourier transform infrared (FT-IR) spectroscopy. Six volunteers ran a marathon race in order to induce changes in their basal plasma FFA levels. Three volunteers were well-trained and three untrained. The non-deconvolved HSA spectra show two types of spectra: the first one includes the untrained runners, who, after the run, display noticeable changes in the absorbance intensity of the amide I band, which also shifts to higher frequencies. The second one includes the well-trained subjects who exhibit few changes after the race. Examination of the deconvolved spectra of HSA shows two types of spectra, as well: the first class displays important decreases in the absorbance of the component assigned to alpha-helix after the race, together with a gradual growth of the two components that can be assigned to turns. The second class, which includes the well-trained subjects, exhibits few changes after the run. These data are interpreted in terms of conformational changes due to a less ordered alpha-helix state of HSA after FFA binding, while the emergence of components assigned to turns may reflect the exposure of several histidine residues to the solvent (which are buried in HSA when FFA/HSA less than 4). Our data are consistent with the Karush model of FFA to HSA binding.

Quantitative study of doxorubicin in living cell nuclei by microspectrofluorometry.

Doxorubicin-DNA association has been studied by quantitative microspectrofluorometry. Fluorescence emission spectra from a microvolume of single living cell nuclei treated with doxorubicin have been analyzed in terms of difference in spectral shape and fluorescence yield between free and DNA-bound drug. Contribution of each spectral component to the total signal was calculated by least-squares linear regression. With this method of analysis, total drug concentration has been determined with an error of less than 10%. Moreover, the uptake into the nucleus has been studied in a non destructive way, avoiding use of 14C-labelled drug. Kinetic studies of drug accumulation into the nuclei were conducted on sensitive and resistant cells.

Structure-activity relation in camptothecin antitumor drugs: why a detailed molecular characterisation of their lactone and carboxylate forms by Raman and SERS spectroscopies?

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.

Monitoring of bacterial growth and structural analysis as probed by FT-IR spectroscopy.

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