Nanocaractérisation par spectroscopies d'émission X et de pertes d'énergie des électrons des réactions physico-chimiques à l'interface verre bioactif / fluide biologique

Cette thèse concerne l'étude des matériaux bioactifs et de leurs propriétés de créer un environnement favorable à la régénération osseuse. Nous nous intéressons à l'étude locale des réactions physico-chimiques précoces se produisant à l'interface verre bioactif / milieu biologique. Les méthodes de caractérisation utilisées sont la microscopie électronique à transmission associée aux spectroscopies de dispersion d'énergie de photons X (EDXS) et de pertes d'énergie des électrons (EELS). Nous avons utilisé la spectrométrie EDXS avec la méthode de quantification de Cliff et Lorimer. Afin d'optimiser les corrections d'absorption pour les éléments légers, nous avons développé un système itéractif de corrections de l'absorption utilisant la valeur expérimentale de l'épaisseur relative locale de l'échantillon mesurée par EELS. Cela a permis de déterminer avec précision les rapports atomiques O/Si dans les différentes zones de l'échantillon. Pour conforter ces mesures, nous avons utilisé l'EELS qui par l'intermédiaire des structures ELNES sur le seuil L2,3 du silicium peut renseigner sur son environnement chimique. Ces deux approches nous ont permis de mettre en évidence l'existence de différentes couches périphériques à l'interface verre bioactif (A9) / fluide biologique (DMEM) : une couche en SiO2, une fine couche temporaire contenant des groupements Si(OH)4, qui permettent la fixation des ions Ca2+. Avec l'arrivée du phospore, nous aboutissons ainsi à la formation d'une couche riche en calcium et en phospore. Au fur et à mesure que cette couche précipite en une apatite, la couche contenant des groupements Si(OH)4 disparaît rapidement par repolymérisation sous la forme SiO2In this thesis we study bioactive materials and their properties to create a favourable environment for the osseous regeneration. More precisely, we are interested in the local and early physicochemical reactions, which occur at the interface between the bioactive glass and the biological environment. The characterisation methods used are transmission electron microscopy associated with energy dispersive X-rays spectroscopy (EDXS) and energy loss electron spectroscopy (EELS). We chose the Cliff and Lorimer method to quantify the elements by EDXS. To optimise the absorption corrections for the light elements, we developed an iterative process for absorption corrections using the experimental value of the local relative specimen thickness measured by EELS. Thus we determined precisely the O/Si atomic ratio in the different areas of the sample. To confirm these measurements, we used EELS to analyse the chemical environment of silicon by the study of the ELNES structures recorded on the L2,3 threshold of silicon. These two approaches allow to demonstrate the existence of many peripheral layers at the bioactive glass (A9) / biological fluid (DMEM) interface: a SiO2 rich layer, a fine temporary layer containing Si(OH)4 groups leading to bonds with calcium ions. Finally by the arrival of phosphorus groups, a calcium and phosphorus rich layer is formed. As this layer precipitates in an apatite, the fine layer containing of the Si(OH)4 groups disappears quickly by repolymerisation in SiO2.REIMS-BU Sciences (514542101) / SudocSudocFranceF

X-ray microanalysis of organic thin sections in TEM using an UTW Si(Li) detector: comparison of quantification methods.

We compared Hall's peak to continuum ratio method with a peak ratio method in order to quantify light elements (C, N, and O) in organic specimens as a model for biological thin sections. X-ray spectra were recorded by an energy dispersive X-ray spectrometer equipped with an ultra thin window detector. Spectra were processed by means of a top-hat filter adapted to peak full-width half maximum. The peak intensities were measured by multiple least square fitting to reference spectra. For most elements of biological interest, theoretical and experimental k-factors were determined. Absorption correction was found to be important for quantitation of carbon, nitrogen, and oxygen. Boron was efficiently detected; however, quantitative analysis was not possible. We conclude from our experiments that the peak ratio method is more suitable for quantitation of elemental concentrations in biological thin sections than the peak to continuum method

Interfacial reactions of glasses for biomedical application by scanning transmission electron microscopy and microanalysis.

récupéré dans HAL-INSERMShort-term physico-chemical reactions at the interface between bioactive glass particles and biological fluids are studied for three glasses with different bioactive properties; these glasses are in the SiO(2)-Na(2)O-CaO-P(2)O(5)-K(2)O-Al(2)O(3)-MgO system. Our aim is to show the difference between the mechanisms of their surface reactions. The relation between the composition and the bioactive properties of these glasses is also discussed. The elemental analysis is performed at the submicrometer scale by scanning transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy. After different immersion times (ranging from 0 to 96 h) of bioactive glass particles in a simulated biological solution, results show the formation of different surface layers at the glass periphery in the case of two bioactive glasses (A9 and BVA). For the third glass (BVH) we do not observe any surface layer formation or any modification of the glass composition. For the two other glasses (A9 and BVA), we observe the presence of different layers: an already observed (Si, O, Al) rich layer at the periphery, a previously demonstrated thin (Si, O) layer formed on top of the (Si, O, Al) layer and a (Ca, P) layer. We determine the different steps of the mechanisms of the surface reactions, which appear to be similar in these glasses, and compare the physico-chemical reactions and kinetics using the different immersion times. The A9 glass permits the observation of all important steps of the surface reactions which lead to bioactivity. This study shows the important relationship between composition and bioactivity which can determine the medical applicability of the glass

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach.

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells

Nanoparticles based on lipidyl-β-cyclodextrins: synthesis, characterization, and experimental and computational biophysical studies for encapsulation of atazanavir

International audienc

Simultaneous imaging of cell shape, mitochondrial potential and nuclear modifications at the onset and during the various stages of apoptosis.

<p>HeLa cells stably expressing H2B-GFP were stained with TMRE to study mitochondrial polarization. Simultaneous time-lapse confocal imaging of cell shape (DIC), TMRE and H2B-GFP was performed by two-photon excitation every five minutes for 7 hours and 15 minutes after the induction of apoptosis by the addition of 500 ng/mL AMD. (A) Traces for TMRE intensity (red line, relative to value reached at time 0.91 h) and nuclear volume (green line, relative to value at time 0 h) in a representative cell (cell #9 on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148727#pone.0148727.s004" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148727#pone.0148727.s006" target="_blank">S3</a> Movies). Mitochondrial depolarization began at 6 h 05 minutes and ended at 6 h 25 minutes, when nuclear volume began to decrease. (B) Cell shape, 3D structure of the TMRE signal, chromatin and nucleus. For each time point, one DIC image (left), one optical section for the red and green signals, a 3D view (surface rendering) of the TMRE signal and a 3D view (surface rendering) of both TMRE signal (red) and H2B-GFP (green) are shown. On DIC image, yellow dotted line indicates the limit of the cell. On 3D view of both TMRE signal (red) and H2B-GFP (green), the relative intensity of the red signal and the volume of the nucleus are indicated by the red and green labels, respectively. Typical chromatin and nucleus structures defined the main stages of apoptosis: stage 1 (ST 1) to stage 5 (ST 5). At the far right of the bottom row, one cell unaffected by AMD after 7 h and 15 minutes is defined as a stage 0 cell (ST 0). In this cell, TMRE staining appears as a 3D network of filaments and the angular nucleus contains a segregated nucleolus. The scale bar represents 10 μm.</p

Simultaneous 3D localization of cytochrome-<i>c</i> (Cc) and H2B-GFP showing Cc redistribution during specific stages of apoptosis.

<p>Anti-cytochrome-<i>c</i> antibody binding was imaged on fixed HeLa cells stably expressing H2B-GFP after the induction of apoptosis by 500 ng/mL AMD, for 7h and 15 minutes. Four images are shown for the same cell, at a given stage. On the first image, differential interference contrast (DIC) shows the shape of the cell, nucleus and nucleolus. The second image is an optical section passing through the middle of the nucleus showing the merge of images for Cc (red) and H2B-GFP (green). The third image is a 3D surface rendering of Cc alone (red). The last image is a simultaneous 3D transparent volume rendering of both Cc (red) and H2B-GFP (green). The scale bar represents 10 μm.</p

“Elemental descriptors” gathering concentration of all the elements/ions considered, for each compartment (condensed chromatin, nucleoplasm, cytosol and mitochondria) in control cells, in cells during each stage of apoptosis and during stage 0.

<p>In the histograms, concentration of nitrogen was divided by 10 (N/10), whereas the concentration of sodium and chloride was multiplied by 3 (Na x 3 and Cl x 3).</p

Targeted quantification of Na⁺, Cl^-, S and Mg²⁺ in the cytosol, mitochondria, condensed chromatin and nucleoplasm.

<p>Concentration of elements/ions (Na⁺, Cl^-, S and Mg²⁺) were determined by energy dispersive X-ray spectrometry in the cytosol, mitochondria, condensed chromatin and nucleoplasm of each of the following: i) control cells, ii) cells in the various stages of apoptosis (ST 1 to ST 5) and iii) cells in the ST 0 stage. Results, in mmol/L, are given as means ±SEM. (<i>n</i> = 3; 3 to 83 different cells per stage).</p