Utilisation de la spectro-imagerie IR-TF pour le développement d'une anatomo-pathologie moléculaire des tumeurs cérébrales

Les gliomes sont des tumeurs agressives de mauvais pronostic, très angiogéniques et infiltrantes ce qui rend leur exérèse particulièrement difficile. Vu les limites des techniques actuelles d imagerie, nous avons proposé la spectro-imagerie Infrarouge à Transformée de Fourier (IRTF), d une résolution spatiale de 6 m, pour apporter une information moléculaire à l examen histologique actuel des gliomes. Nos travaux ont été fondés sur la recherche de paramètres moléculaires des vaisseaux sanguins, notamment sur la base des contenus de leur membrane basale. Celle-ci subit des altérations dûes au stress angiogénique tumoral. Nous avons mis en évidence des altérations de la structure secondaire des protéines (tels les collagènes) des vaisseaux sanguins au cours de la croissance de la tumeur. Nous avons aussi évalué les modifications des chaines d acides gras des phospholipides membranaires, qui révélent un degré d insaturation plus important pour les vaisseaux tumoraux. Ensuite, sur un modèle de gliome murin, nous avons établi une méthode efficace de classification des capillaires sanguins sur la base d absorptions de leurs contenus glucidiques et lipidiques, permettant de discriminer totalement les capillaires sains et tumoraux. La combinaison de ces paramètres a été mise à profit pour assurer une histopathologie moléculaire des gliomes humains. Nos résultats ont démontré qu il est possible de différencier entre la vasculature saine et tumorale sur ces gliomes humains, ce qui permet une bonne délimitation des zones tissulaires correspondantes. Cette technique pourrait devenir un outil analytique fiable, rapide d une durée compatible avec la chirurgie et donc très utile pour les neurochirurgiens.Malignant gliomas are very aggressive tumors with poor prognosis, highly angiogenic and invasive into the surrounding brain parenchyma, making their resection very difficult. Regarding the limits of current imaging techniques, we have proposed Fourier Transform Infrared (FTIR) spectro-imaging, with a spatial resolution of 6 m, to provide molecular information for the histological examination of gliomas. Our work was based on the research of molecular parameters of blood vessels, notably on the basis of the contents of their basement membrane, which undergoes changes due to tumor angiogenic stress. We have identified alterations of the secondary structure of proteins (such as collagen) in blood vessels during tumor growth. We have also assessed the changes in fatty acyl chains of membrane phospholipids, which revealed a higher unsaturation level in tumor vessels. Then, on a murine glioma model, we have established an efficient method of blood vessels classification based on their carbohydrates and fats contents, allowing the differentiation between healthy and tumor blood vessels. The combination of these parameters was used to provide a molecular histopathology for the study of human gliomas. Our results have demonstrated the feasibility of differentiating between healthy and tumor vasculature in these human gliomas, which help delimitating areas of corresponding tissue. This technique could become a reliable and fast analytical tool, with duration compatible with the surgery and thus very useful for neurosurgeons.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

The effect of optical substrates on micro-FTIR analysis of single mammalian cells

The study of individual cells with infrared (IR) microspectroscopy often requires living cells to be cultured directly onto a suitable substrate. The surface effect of the specific substrates on the cell growth—viability and associated biochemistry—as well as on the IR analysis—spectral interference and optical artifacts—is all too often ignored. Using the IR beamline, MIRIAM (Diamond Light Source, UK), we show the importance of the substrate used for IR absorption spectroscopy by analyzing two different cell lines cultured on a range of seven optical substrates in both transmission and reflection modes. First, cell viability measurements are made to determine the preferable substrates for normal cell growth. Successively, synchrotron radiation IR microspectroscopy is performed on the two cell lines to determine any genuine biochemically induced changes or optical effect in the spectra due to the different substrates. Multivariate analysis of spectral data is applied on each cell line to visualize the spectral changes. The results confirm the advantage of transmission measurements over reflection due to the absence of a strong optical standing wave artifact which amplifies the absorbance spectrum in the high wavenumber regions with respect to low wavenumbers in the mid-IR range. The transmission spectra reveal interference from a more subtle but significant optical artifact related to the reflection losses of the different substrate materials. This means that, for comparative studies of cell biochemistry by IR microspectroscopy, it is crucial that all samples are measured on the same substrate type. [Figure: see text

Rapid recognition of drug-resistance/sensitivity in leukemic cells by Fourier transform infrared microspectroscopy and unsupervised hierarchical cluster analysis

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Conduit Dynamics in Transitional Rhyolitic Activity Recorded by Tuffisite Vein Textures from the 2008–2009 Chaitén Eruption

The mechanisms of hazardous silicic eruptions are controlled by complex, poorly-understood conduit processes. Observations of recent Chilean rhyolite eruptions have revealed the importance of hybrid activity, involving simultaneous explosive and effusive emissions from a common vent. Such behavior hinges upon the ability of gas to decouple from magma in the shallow conduit. Tuffisite veins are increasingly suspected to be a key facilitator of outgassing, as they repeatedly provide a transient permeable escape route for volcanic gases. Intersection of foam domains by tuffisite veins appears critical to efficient outgassing. However, knowledge is currently lacking into textural heterogeneities within shallow conduits, their relationship with tuffisite vein propagation, and the implications for fragmentation and degassing processes. Similarly, the magmatic vesiculation response to upper conduit pressure perturbations, such as those related to the slip of dense magma plugs, remains largely undefined. Here we provide a detailed characterization of an exceptionally large tuffisite vein within a rhyolitic obsidian bomb ejected during transitional explosive-effusive activity at Chaitén, Chile in May 2008. Vein textures and chemistry provide a time-integrated record of the invasion of a dense upper conduit plug by deeper fragmented magma. Quantitative textural analysis reveals diverse vesiculation histories of various juvenile clast types. Using vesicle size distributions, bubble number densities, zones of diffusive water depletion, and glass H2O concentrations, we propose a multi-step degassing/fragmentation history, spanning deep degassing to explosive bomb ejection. Rapid decompression events of ~3–4 MPa are associated with fragmentation of foam and dense magma at ~200–360 m depth in the conduit, permitting vertical gas and pyroclast mobility over hundreds of meters. Permeable pathway occlusion in the dense conduit plug by pyroclast accumulation and sintering preceded ultimate bomb ejection, which then triggered a final bubble nucleation event. Our results highlight how the vesiculation response of magma to decompression events is highly sensitive to the local melt volatile concentration, which is strongly spatially heterogeneous. Repeated opening of pervasive tuffisite vein networks promotes this heterogeneity, allowing juxtaposition of variably volatile-rich magma fragments that are derived from a wide range of depths in the conduit. This process enables efficient but explosive removal of gas from rhyolitic magma and creates a complex textural collage within dense rhyolitic lava, in which neighboring fused clasts may have experienced vastly different degassing histories

Label-free characterization of biochemical changes within human cells under parasite attack using synchrotron based micro-FTIR

© 2019 The Royal Society of Chemistry. The protozoan Toxoplasma gondii is responsible for severe, potentially life-threatening, infection in immunocompromised individuals and when acquired during pregnancy. In the meantime, there is no available vaccine and the anti-T. gondii drug arsenal is limited. An important challenge to improve antiparasitic therapy is to understand chemical changes that occur during infection. Here, we used Fourier transform infrared spectroscopy (FTIR) to investigate the effect of T. gondii infection on the chemical composition of human brain microvascular endothelial cells (hBMECs) at 3, 6, 24 and 48 hours postinfection (hpi). Principal component analysis (PCA) showed that the best separation and largest difference between infected and uninfected hBMECs was detected at 24 hpi and within the 3400-2800 cm-1 region. At 48 hpi, although the difference between samples was obvious within the 3400-2800 cm-1 region, more differences were detected in the fingerprint region. These findings indicate that infected and control cells can be easily distinguished. Although differences between the spectra varied, the separation was most clear at 24 hpi. T. gondii increased signals for lipids (2853 cm-1) and nucleic acids (976 cm-1, 1097 cm-1 and 1245 cm-1), and decreased signals for proteins (3289 cm-1, 2963 cm-1, 2875 cm-1) in infected cells compared to controls. These results, supported by amino acid levels in culture media, and global metabolomic and gene expression analyses of hBMECs, suggest that T. gondii parasite exploits a wide range of host-derived chemical compounds and signaling pathways for its own survival and proliferation within host cells. Our data demonstrate that FTIR combined with chemometric analysis is a valuable approach to elucidate the temporal, infection-specific, chemical alterations in host cells at a single cell resolution

Discrimination between two different grades of human glioma based on blood vessel infrared spectral imaging

Gliomas are brain tumours classified into four grades with increasing malignancy from I to IV. The development and the progression of malignant glioma largely depend on the tumour vascularization. Due to their tissue heterogeneity, glioma cases can be difficult to classify into a specific grade using the gold standard of histological observation, hence the need to base classification on a quantitative and reliable analytical method for accurately grading the disease. Previous works focused specifically on vascularization study by Fourier transform infrared (FTIR) spectroscopy, proving this method to be a way forward to detect biochemical changes in the tumour tissue not detectable by visual techniques. In this project, we employed FTIR imaging using a focal plane array (FPA) detector and globar source to analyse large areas of glioma tumour tissue sections via molecular fingerprinting in view of helping to define markers of the tumour grade. Unsupervised multivariate analysis (hierarchical cluster analysis and principal component analysis) of blood vessel spectral data, retrieved from the FPA images, revealed the fine structure of the borderline between two areas identified by a pathologist as grades III and IV. Spectroscopic indicators are found capable of discriminating different areas in the tumour tissue and are proposed as biomolecular markers for potential future use of grading gliomas. Graphical Abstract Infrared imaging of glioma blood vessels provides a means to revise the pathologists' line of demarcation separating grade III (GIII) from grade IV (GIV) parts

Usage of FTIR spectro-imaging for the development of a molecular anatomo-pathology of cerebral tumors

Les gliomes sont des tumeurs agressives de mauvais pronostic, très angiogéniques et infiltrantes ce qui rend leur exérèse particulièrement difficile. Vu les limites des techniques actuelles d’imagerie, nous avons proposé la spectro-imagerie Infrarouge à Transformée de Fourier (IRTF), d’une résolution spatiale de 6 µm, pour apporter une information moléculaire à l’examen histologique actuel des gliomes. Nos travaux ont été fondés sur la recherche de paramètres moléculaires des vaisseaux sanguins, notamment sur la base des contenus de leur membrane basale. Celle-ci subit des altérations dûes au stress angiogénique tumoral. Nous avons mis en évidence des altérations de la structure secondaire des protéines (tels les collagènes) des vaisseaux sanguins au cours de la croissance de la tumeur. Nous avons aussi évalué les modifications des chaines d’acides gras des phospholipides membranaires, qui révélent un degré d’insaturation plus important pour les vaisseaux tumoraux. Ensuite, sur un modèle de gliome murin, nous avons établi une méthode efficace de classification des capillaires sanguins sur la base d’absorptions de leurs contenus glucidiques et lipidiques, permettant de discriminer totalement les capillaires sains et tumoraux. La combinaison de ces paramètres a été mise à profit pour assurer une histopathologie moléculaire des gliomes humains. Nos résultats ont démontré qu’il est possible de différencier entre la vasculature saine et tumorale sur ces gliomes humains, ce qui permet une bonne délimitation des zones tissulaires correspondantes. Cette technique pourrait devenir un outil analytique fiable, rapide d’une durée compatible avec la chirurgie et donc très utile pour les neurochirurgiens.Malignant gliomas are very aggressive tumors with poor prognosis, highly angiogenic and invasive into the surrounding brain parenchyma, making their resection very difficult. Regarding the limits of current imaging techniques, we have proposed Fourier Transform Infrared (FTIR) spectro-imaging, with a spatial resolution of 6 µm, to provide molecular information for the histological examination of gliomas. Our work was based on the research of molecular parameters of blood vessels, notably on the basis of the contents of their basement membrane, which undergoes changes due to tumor angiogenic stress. We have identified alterations of the secondary structure of proteins (such as collagen) in blood vessels during tumor growth. We have also assessed the changes in fatty acyl chains of membrane phospholipids, which revealed a higher unsaturation level in tumor vessels. Then, on a murine glioma model, we have established an efficient method of blood vessels classification based on their carbohydrates and fats contents, allowing the differentiation between healthy and tumor blood vessels. The combination of these parameters was used to provide a molecular histopathology for the study of human gliomas. Our results have demonstrated the feasibility of differentiating between healthy and tumor vasculature in these human gliomas, which help delimitating areas of corresponding tissue. This technique could become a reliable and fast analytical tool, with duration compatible with the surgery and thus very useful for neurosurgeons

Utilisation de la spectro-imagerie IR-TF pour le développement d'une anatomo-pathologie moléculaire des tumeurs cérébrales

Les gliomes sont des tumeurs agressives de mauvais pronostic, très angiogéniques et infiltrantes ce qui rend leur exérèse particulièrement difficile. Vu les limites des techniques actuelles d’imagerie, nous avons proposé la spectro-imagerie Infrarouge à Transformée de Fourier (IRTF), d’une résolution spatiale de 6 µm, pour apporter une information moléculaire à l’examen histologique actuel des gliomes. Nos travaux ont été fondés sur la recherche de paramètres moléculaires des vaisseaux sanguins, notamment sur la base des contenus de leur membrane basale. Celle-ci subit des altérations dûes au stress angiogénique tumoral. Nous avons mis en évidence des altérations de la structure secondaire des protéines (tels les collagènes) des vaisseaux sanguins au cours de la croissance de la tumeur. Nous avons aussi évalué les modifications des chaines d’acides gras des phospholipides membranaires, qui révélent un degré d’insaturation plus important pour les vaisseaux tumoraux. Ensuite, sur un modèle de gliome murin, nous avons établi une méthode efficace de classification des capillaires sanguins sur la base d’absorptions de leurs contenus glucidiques et lipidiques, permettant de discriminer totalement les capillaires sains et tumoraux. La combinaison de ces paramètres a été mise à profit pour assurer une histopathologie moléculaire des gliomes humains. Nos résultats ont démontré qu’il est possible de différencier entre la vasculature saine et tumorale sur ces gliomes humains, ce qui permet une bonne délimitation des zones tissulaires correspondantes. Cette technique pourrait devenir un outil analytique fiable, rapide d’une durée compatible avec la chirurgie et donc très utile pour les neurochirurgiens.Malignant gliomas are very aggressive tumors with poor prognosis, highly angiogenic and invasive into the surrounding brain parenchyma, making their resection very difficult. Regarding the limits of current imaging techniques, we have proposed Fourier Transform Infrared (FTIR) spectro-imaging, with a spatial resolution of 6 µm, to provide molecular information for the histological examination of gliomas. Our work was based on the research of molecular parameters of blood vessels, notably on the basis of the contents of their basement membrane, which undergoes changes due to tumor angiogenic stress. We have identified alterations of the secondary structure of proteins (such as collagen) in blood vessels during tumor growth. We have also assessed the changes in fatty acyl chains of membrane phospholipids, which revealed a higher unsaturation level in tumor vessels. Then, on a murine glioma model, we have established an efficient method of blood vessels classification based on their carbohydrates and fats contents, allowing the differentiation between healthy and tumor blood vessels. The combination of these parameters was used to provide a molecular histopathology for the study of human gliomas. Our results have demonstrated the feasibility of differentiating between healthy and tumor vasculature in these human gliomas, which help delimitating areas of corresponding tissue. This technique could become a reliable and fast analytical tool, with duration compatible with the surgery and thus very useful for neurosurgeons

Tracking calcification in tissue-engineered bone using synchrotron micro-FTIR and SEM

One novel tissue engineering approach to mimic in vivo bone formation is the use of aggregate or micromass cultures. Various qualitative and quantitative techniques, such as histochemical staining, protein assay kits and RT-PCR, have been used previously on cellular aggregate studies to investigate how these intricate arrangements lead to mature bone tissue. However, these techniques struggle to reveal spatial and temporal distribution of proliferation and mineralization simultaneously. Synchrotron-based Fourier transform infrared microspectroscopy (micro-FTIR) offers a unique insight at the molecular scale by coupling high IR sensitivity to organic matter with the high spatial resolution allowed by diffraction limited SR microbeam. This study is set to investigate the effects of culture duration and aggregate size on the dynamics and spatial distribution of calcification in engineered bone aggregates by a combination of micro-FTIR and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDX). A murine bone cell line has been used, and small/large bone aggregates have been induced using different chemically treated culture substrates. Our findings suggest that bone cell aggregate culturing can greatly increase levels of mineralization over short culture periods. The size of the aggregates influences mineralisation rates with larger aggregates mineralizing at a faster rate than their smaller counterparts. The micro-FTIR mapping has demonstrated that mineralization in the larger aggregates initiated from the periphery and spread to the centre, whilst the smaller aggregates have more minerals in the centre at the early stage and deposited more in the periphery after further culturing, implying that aggregate size influences calcification distribution and development over time. SEM/EDX data correlates well with the micro-FTIR results for the total mineral content. Thus, synchrotron-based micro-FTIR can accurately track mineralization process/mechanism in the engineered bone