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International audience; High dose selenium acts as a cytotoxic agent, with potential applications in cancer treatment. However, clinical trials have failed to show any chemotherapeutic value of selenium at safe and tolerated doses (

Advanced science xx, 2001675 (1-18) (2020). doi:10.1002/advs.202001675, To improve the prognosis of glioblastoma, innovative radiotherapy regimens are required to augment the effect of tolerable radiation doses while sparing surrounding tissues. In this context, nanoscintillators are emerging radiotherapeutics that down‐convert X‐rays into photons with energies ranging from UV to near‐infrared. During radiotherapy, these scintillating properties amplify radiation‐induced damage by UV‐C emission or photodynamic effects. Additionally, nanoscintillators that contain high‐Z elements are likely to induce another, currently unexplored effect: radiation dose‐enhancement. This phenomenon stems from a higher photoelectric absorption of orthovoltage X‐rays by high‐Z elements compared to tissues, resulting in increased production of tissue‐damaging photo‐ and Auger electrons. In this study, Geant4 simulations reveal that rare‐earth composite LaF$_3$:Ce nanoscintillators effectively generate photo‐ and Auger‐electrons upon orthovoltage X‐rays. 3D spatially resolved X‐ray fluorescence microtomography shows that LaF$_3$:Ce highly concentrates in microtumors and enhances radiotherapy in an X‐ray energy‐dependent manner. In an aggressive syngeneic model of orthotopic glioblastoma, intracerebral injection of LaF$_3$:Ce is well tolerated and achieves complete tumor remission in 15% of the subjects receiving monochromatic synchrotron radiotherapy. This study provides unequivocal evidence for radiation dose‐enhancement by nanoscintillators, eliciting a prominent radiotherapeutic effect. Altogether, nanoscintillators have invaluable properties for enhancing the focal damage of radiotherapy in glioblastoma and other radioresistant cancers., Published by Wiley-VCH, Weinheim

International audience; To better understand the physiology and acclimation capability of the cell, one of the great challenges of the future is to access the interior of a cell and unveil its chemical landscape (composition and distribution of elements and molecules). Chemical imaging has greatly improved in sensitivity and spatial resolution to visualize and quantify nutrients, metabolites, toxic elements, and drugs in single cells at the subcellular level. This review aims to present the current potential of these emerging imaging technologies and to guide biologists towards a strategy for interrogating biological processes at the nanoscale. We also describe various solutions to combine multiple imaging techniques in a correlative way and provide perspectives and future directions for integrative subcellular imaging across different disciplines

Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson’s disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson’s disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.

International audience; Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm  50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.

International audience; We have recently developed a series of tamoxifen-like metallocifens of the group-8 metals (Fe, Ru and Os) with strong antiproliferative activity on the triple negative breast cancer cells (MDA-MB-231). This property, not observed for the organic analog 4-hydroxytamoxifen, has been associated with the unique redox behavior of metallocenic moieties, readily affording reactive quinone methides in cancer cells. To shed light on the mechanism of action of these molecules, synchrotron radiation X-ray fluorescence (SR-XRF) nanoimaging studies were performed on cells exposed to osmocenyltamoxifen (Oc-OH-Tam) to disclose its intracellular distribution using osmium as an intrinsic reporter. High resolution mapping of the lipophilic Oc-OH-Tam in cells, revealed its preferential accumulation in the endomembrane system encompassing endoplasmic reticulum,nuclear envelope and vesicular structures. This is consistent with the ability of the amino nitrogen chain of the compounds to be protonated at physiological pH and responsible for electrostatic interactions between Oc-OH-Tam and membranes. We propose a comprehensive scenario that provides new insight into the cellular behavior and activation of Oc-OH-Tam and advances the understanding of its mechanism of action.

Standard histopathological examination is the gold standard for many disease diagnoses although the technique is limited with no full 3D volume analysis possible. Three dimensional X-ray Phase-Contrast Imaging(PCI) methods have been under constant and fast developments in the recent decades due to their superior performance for imaging low density objects and their ability to provide complementary information compared to attenuation based imaging. Despite the progresses, X-ray Phase Contrast Tomography still encounters remaining challenges to overcome on its way to become a routine non-invasive technique allowing the 3D assessment of tissue architecture in laboratory set-ups. Speckle Based Imaging (SBI) forms a new class of X-ray PCI techniques, sensitive to the first derivative of the phase. The set-up involved and the simplicity of implementation provide many advantages to SBI such as having no field of view and no resolution limitation in addition to have low requirements on the beam coherences. These advantages make SBI a good candidate for the transfer on conventional sources. In this work, we present preliminary results obtained on a conventional μCT and their comparison with data acquired at the European Synchrotron. We used a new phase retrieval algorithm based on optical energy conservation. We applied the method on both phantoms and biological samples in order to evaluate its quantitativeness for a transfer. A comparison to previously available speckle tracking algorithms is also performed. We demonstrate that the combination of the phase retrieval method with a standard μCT can achieve high resolution and high contrast within a few minutes, with a comparable image quality to the results obtained using synchrotron light.

International audience; The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (μg/kg) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.

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International audience; Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles' ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.

International audience; The ID16A beamline at ESRF offers unique capabilities for X-ray nano-imaging, and currently produces the worlds brightest high energy diffraction-limited nanofocus. Such a nanoprobe was designed for quantitative characterization of the morphology and the elemental composition of specimens at both room and cryogenic temperatures. Billions of photons per second can be delivered in a diffraction-limited focus spot size down to 13 nm. Coherent X-ray imaging techniques, as magnified holographic-tomography and ptychographic-tomography, are implemented as well as X-ray fluorescence nanoscopy. We will show the latest developments in coherent and spectroscopic X-ray nanoimaging implemented at the ID16A beamline.

International audience; Parkinson’s disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson’s disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson’s disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

Manganese enhanced MRI (MEMRI) offers many possibilities such as tract tracing of neuronal pathways and functional imaging in vivo. This technique necessitates a direct or indirect acute injection of MnCl2 in the brain. Unfortunately, local concentrations of Mn2+ and its impact on metabolism after a single injection remain largely unknown. In this study, we combined in vivo MEMRI and ex vivo Proton High Resolution Magic Angle Spinning MRS (1H HRMAS MRS) to investigate the delayed impact of Mn2+ on rat hippocampal metabolism. MEMRI images were acquired 24 h after MnCl2 injection in the dentate gyrus of the rat hippocampus at two different Mn2+ doses: low (8 nmol; n = 16) and high (500 nmol; n = 10). The low Mn2+ dose had almost no impact on hippocampal metabolism while the high dose altered the amplitude of several metabolites (up to +54% for Glu and −71% for Asp). Moreover, at this high dose, the paramagnetic properties of Mn2+ led to a broadening of the resonances of several organic acids (lactate, glutamate, N-acetyl aspartate etc.), suggesting a chelation of Mn2+ and an impact on Mn2+ relaxivity. Metabolite amplitudes were well correlated with the Mn2+ concentrations measured with an MRI T1-map (glutamate: R2 = 0.8, p = 0.02; phosphoethanolamine: R2 = 0.9, p = 0.0004; γ-aminobutyrate: R2 = 0.7, p = 0.005 and phosphocholine: R2 = 0.6, p = 0.04). To conclude, HRMAS is well suited to investigate Mn2+ impact on the metabolism. The low Mn2+ dose (8 nmol) usually used in the MEMRI experiment does not impact the hippocampal metabolism. The chelation of Mn2+ and its impact on relaxivity suggests an over-estimation of the Mn2+ concentration when measured through a T1 map.

International audience; The MAP6 (microtubule-associated protein 6) KO mouse is a microtubule-deficient model of schizophrenia that exhibits severe behavioral disorders that are associated with synaptic plasticity anomalies. These defects are alleviated not only by neuroleptics, which are the gold standard molecules for the treatment of schizophrenia, but also by Epothilone D (Epo D), which is a microtubule-stabilizing molecule. To compare the neuronal transport between MAP6 KO and wild-type mice and to measure the effect of Epo D treatment on neuronal transport in KO mice, MnCl2 was injected in the primary somatosensory cortex. Then, using manganese-enhanced magnetic resonance imaging (MEMRI), we followed the propagation of Mn(2+) through axonal tracts and brain regions that are connected to the somatosensory cortex. In MAP6 KO mice, the measure of the MRI relative signal intensity over 24h revealed that the Mn(2+) transport rate was affected with a stronger effect on long-range and polysynaptic connections than in short-range and monosynaptic tracts. The chronic treatment of MAP6 KO mice with Epo D strongly increased Mn(2+) propagation within both mono- and polysynaptic connections. Our results clearly indicate an in vivo deficit in neuronal Mn(2+) transport in KO MAP6 mice, which might be due to both axonal transport defects and synaptic transmission impairments. Epo D treatment alleviated the axonal transport defects, and this improvement most likely contributes to the positive effect of Epo D on behavioral defects in KO MAP6 mice.

International audience; The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the nucleus at high concentration. Exposure to low concentrations of cobalt or silver did not alter the localization nor the concentration of endogenous elements within the cells. To our knowledge, this is the first report on element co-localization and segregation at the sub-cellular level in micro-algae by means of synchrotron nano X-ray fluorescence spectroscopy.

International audience; Root cell walls accumulate metal cations both during acquisition from the environment and removal from the protoplast to avoid toxicity, but molecular forms of the metals under field conditions remain elusive. We have identified how copper is bound to cell walls of intact roots of native Thlaspi arvense by combining synchrotron X-ray fluorescence and absorption techniques (XANES and EXAFS) at the nano-, micro-, and bulk scales. The plants grew naturally in sediment in a stormwater runoff basin at copper concentrations typical of urban ecosystems. About 90% of acquired copper is bound in vivo to cell walls as a unique five-coordinate Cu(II)-bis(L-histidinato) complex with one L-histidine behaving as a tridentate ligand (histamine-like chelate) and the other as a bidentate ligand (glycine-like chelate). Tridentate binding of Cu(II) would provide thermodynamic stability to protect cells against copper toxicity, and bidentate binding may enable kinetic lability along the cell wall through protein-protein docking with the non-bonded imidazole group of histidine residues. EXAFS spectra are provided as ESI dagger to facilitate further identification of Cu-histidine and distinction of Cu-N from Cu-O bonds in biomolecules.

International audience; Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected zinc mass fraction in whole cell of PC12 cell lines. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements while quantitative phase contrast imaging provides maps of the projected mass. The combined method was validated on calibration samples by comparison with other alternative techniques such as Atomic Force Microscopy (AFM) and Scanning Transmission Ion Microscopy (STIM). Up to now, absolute quantification at the sub-cellular level was impossible using X-ray fluorescence microscopy but can be reached for the first time with the use of the proposed approach

International audience; Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).

International audience; AIMS: In cancer therapy, research has focused on the development of nanocarriers that can aid diagnosis, deliver therapeutic agents and monitor treatment progress. This study introduces high-resolution synchrotron x-ray fluorescence microscopy (SR-XFM) to investigate intracellular localization of novel lanthanide-coated nanoparticles in human cells and their genotoxicity screening after internalization. MATERIALS & METHODS: Noble metal nanoparticles coated with cerium and luminescent europium complexes have been developed as platforms for bioimaging and potential biodelivery applications. The intracellular distribution after internalization has been analyzed by ultrasensitive SR-XFM and genotoxicity evaluated using γ-H2AX DNA damage foci phosphorylation assay. RESULTS: We demonstrate the unprecedented capability of SR-XFM for extremely sensitive nanoimaging and intracellular elemental distribution analysis of noble metal nanoparticles in cells. Furthermore, we show that, depending on the charge of the coating complex and the presence of the DNA cargo, the internalization of functionalized nanoparticles by human fibroblasts can cause elevated levels of DNA damage detected by histone H2AX phosphorylation. CONCLUSION: The variable genotoxic impact of newly designed nanovectors emphasizes the need for careful and comprehensive testing of biological responses of all new nanoconstructs intended for future clinical applications. This can be greatly facilitated by SR-XFM nanoimaging of nanoparticles in cells at very low concentrations.

International audience; Chronic exposure to manganese results in neurological symptoms referred to as manganism and is identified as a risk factor for Parkinson's disease. In vitro, manganese induces cell death in the dopaminergic cells, but the mechanisms of manganese cytotoxicity are still unexplained. In particular, the subcellular distribution of manganese and its interaction with other trace elements needed to be assessed. Applying synchrotron X-ray fluorescence nanoimaging, we found that manganese was located within the Golgi apparatus of PC12 dopaminergic cells at physiologic concentrations. At increasing concentrations, manganese accumulates within the Golgi apparatus until cytotoxic concentrations are reached resulting in a higher cytoplasmic content probably after the Golgi apparatus storage capacity is exceeded. Cell exposure to manganese and brefeldin A, a molecule known to specifically cause the collapse of the Golgi apparatus, results in the striking intracellular redistribution of manganese, which accumulates in the cytoplasm and the nucleus. These results indicate that the Golai apparatus plays an important role in the cellular detoxification of manganese. In addition manganese exposure induces a decrease in total iron content, which could contribute to the overall neurotoxicity.

The X-ray Microscopy and Micro-analysis beamlines at ESRF operate complementary state-of-the-art instruments at ID21, ID22, ID18F and more recently ID22NI. Within a multi-modal strategy, these beamlines develop micro-imaging techniques with various contrast mechanisms (μXRF, μXANES, μXRD and phase contrast) and host experiments with scientific topics ranging from Geochemistry to Archeology, Environmental sciences, Biology and Material sciences. Future challenges include pushing spatial resolution down to the nano-scale and the development of innovative 3D micro-analysis techniques.

International audience; The microchemical environment of neuromelanin (NM) in whole neurons from formalin fixed and paraffin embedded human substantia nigra sections were characterized using synchrotron chemical X-ray microscopy. Concentrations of NM-associated elements increased in the developing brain; the highest levels of most elements were found in the mature brain but the temporal pattern of the accumulation of different elements varied. High spatial resolution investigations, using a unique hard X-ray nanoprobe, revealed iron-rich microdomains colocalized with other elements within the pigment. These microdomains represent the first visualization of a structure regulating the metal-binding properties of NM and supporting a physiological role for NM in the regulation of functionally important elements in pigmented neurons. Our results demonstrate that the local chemical environment of iron in NM is similar to that found in ferritin and points to a possible role of iron in NM biosynthesis. Intracellular speciation of sulfur contained in NM revealed the presence of reduced sulfur compounds and various forms of oxidized sulfur compounds which have not previously been reported. Further, a significant increase in sulfonate in NM in the mature brain suggests that in vivo metabolism of the pigment via an as yet unidentified pathway occurs. The current data add to our understanding of the development and regulation of NM in the human brain.

International audience; The causes of Parkinson's disease and amyotrophic lateral sclerosis are still not known, but there is evidence that metal ions can be involved in processes leading to degeneration and atrophy of neurons in the case of these two neurodegenerative disorders. A synchrotron microbeam X-ray fluorescence technique was applied for topographic and quantitative analyses of selected elements on central nervous system tissue. The thin slices of brain were measured on the undulator beamline ID 22 at the European Synchrotron Radiation Facility in Grenoble, France. The polychromatic beam with the dimension of 5 microm x 2 microm (horizontal x vertical) was used in measurements. Tissues of substantia nigra representing Parkinson's disease, amyotrophic lateral sclerosis, and the control case were scanned. The results obtained indicated that accumulation of some elements depends on the case that the substantia nigra represents. Some variability in the elemental distribution for a given case was noticed as well. To investigate if present differences in the elemental accumulation between analyzed cases are statistically significant, multivariate methods were used. Cluster and discriminant analyses confirmed the significance of the differences in elemental accumulation in biological structures representing the examined cases. The methods used let us classify these structures in separate groups and determine elements, which play the greatest role in the differentiation of the biological structures for each case.

Conventional radiotherapy of high-grade glioma is unsuccessful since less than 50% of patients survive at 6 months, therefore glioma treatment is still challenging. A new radiotherapy procedure has been recently proposed, the photoactivation therapy (PAT), associating synchrotron radiation with a chemotherapy agent, such as cisplatin. PAT aims at using the monochromaticity and the very high brilliance of the synchrotron radiation for selective excitation of a high-Z compound introduced in tumor cell DNA to maximize the photoelectric effect probability, thus increasing local toxicity. Synchrotron irradiation of cisplatin at the platinum absorption K-edge resulted in a dramatic increase in life span relative to median survival time in the F98 glioma model in Fisher rat. In the purpose to optimize the platinum concentration into the tumor, the platinum content of irradiated target needs to be quantified. These results will enable to correlate injected dose to cellular platinum content in the tumor at the time of irradiation, and to study the spatial diffusion and distribution of the platinum into the tumor and the surrounding healthy tissues from the point of injection. Male Fisher 344 rats were inoculated with 103 F98 glioma cells. Thirteen days after stereotactic inoculation, intracerebral injection at the tumor site of 40 μg of carboplatin and 3 or 5 μg of cisplatin was performed. Platinum quantitative distribution in tumors and adjacent brain tissues was determined using μ-PIXE and μ-RBS analysis.

High-grade gliomas are usually of poor prognosis, and conventional radiotherapy, even combined with chemotherapy, still fails to improve the survival of patients. Here, we propose an innovative therapeutic approach combining synchrotron radiation with cis-diamminedichloroplatinum (II) (CDDP). As suggested previously, monochromatic synchrotron irradiation of CDDP at 78.8 keV, just above the 78.4 keV platinum absorption K-edge, leads to an enhanced photoelectric effect and an increased local toxicity. To select a particular radiation energy that could provide supra-additive effect, we used pulsed-field gel electrophoresis to assess yields of DNA double-strand breaks induced in rat F98 glioma cells after CDDP treatment combined with synchrotron X-rays. Thereafter, intracerebral CDDP injection combined with synchrotron X-rays was applied to Fisher rats bearing F98 glioma. CDDP concentrations were mapped by synchrotron X-ray microfluorescence. An extra number of more slowly repaired double strand breaks were observed when irradiating CDDP-treated F98 cells at 78.8 keV. In vivo treatments were then performed with different radiation doses and CDDP concentrations. All cell inoculations in rat brain resulted in tumor development, and tumor presence was controlled by computed tomography. Among all of the conditions tested, the combination of 3 μg of CDDP with 15 Gy resulted in the largest median survival time (206 days). After 1 year, about 34% of treated rats were still alive. This preclinical finding, validated by molecular analysis, represents the most protracted survival reported with this radioresistant glioma model and demonstrates the interest in powerful monochromatic X-ray sources as new tools for cancer treatments.

A feasibility study of previous termquantitativenext term elemental microanalysis of biological materials and glass samples by microbeam X-ray fluorescence spectroscopy was completed. The previous termresearchnext term included testing the homogeneity of existing standards for X-ray fluorescence calibration and verification of a fundamental parameters method for previous termquantitativenext term analysis. The goal was to evaluate the X-ray fluorescence spectrometer as a tool for elemental analysis at the microscale level. Glass Standard Reference Materials were analyzed. The glass specimens consisted of flat, optically polished slabs having three different thicknesses. For calibration, metal thin films were used. The microbeam X-ray fluorescence spectrometer utilizing capillary optics with effective beam diameter equal to about 30 μm has been applied previous termin this research.next term Sources of uncertainties considered previous terminnext term this work were detector and X-ray tube stability, specimen movement, and spectral deconvolution. Concentrations of analytes were calculated using a fundamental parameters approach. Coherently and incoherently scattered lines of tube target were used for matrix correction and to estimate the mass thickness of the sample. The synchrotron microbeam X-ray fluorescence technique was used for previous termquantitativenext term analysis of human brain tissue samples. previous termInnext term measurements the monochromatic and polychromatic synchrotron microbeams were applied. The same area of tissue sample was scanned with the use of both X-ray microbeams. The concentrations of selected elements were computed. A reasonably good agreement between results of both analyses was obtained

International audience; The aim of this study was to establish the feasibility of obtaining 3D elemental images of fluid inclusions, which consist of multiphase and irregular objects trapped in a host mineral. These experiments are based on the fluorescence micro-tomography technique, which combines X-ray fluorescence measurements and 2D tomographic reconstruction algorithms. For this purpose, multiphase aqueous fluid inclusions were synthesized in fractured quartz crystal within gold capsule at high temperature and pressure. A single fluid inclusion was isolated in a tiny quartz parallelepiped, polished on all its faces. On the basis of the parallel collection technique, the sample was successively scanned vertically over a few hundred micrometers, in steps of 3 μm and with an acquisition time of 1 s/point, and rotated by an angle of 2°. The 2D projection images, which consist in relative elemental concentration regions, provide the size and the location of the fluid inclusion as well as the elemental location and combination. By iterating the procedure along the horizontal rotation axis, full 3D elemental images can be obtained.

Hydroxypropylmethylcellulose (HPMC) of high-viscosity grade is used as a ligand for a bioactive calcium phosphate ceramic (the filler) in a ready-to-use injectable sterilized biomaterial for bone and dental surgery. Application of physico-chemical methods such as XPS, NMR, or Raman spectroscopy encounters difficulties when used to study such a multiphased material. This paper reports on the application of FT-IR microspectroscopy (FT-IRM) for the investigation of inorganic and organic phases of the rough composite and separated phases obtained by mechanical or chemical extraction methods. A comparison of FT-IRM with the conventional KBr pellet method was made and indicates that the macro and micro FT-IR methods are complementary: the former revealed new chemical groups not visualized with the KBr method whereas the latter detected the major compound of the blend. FT-IR microspectroscopy was revealed to be a powerful method of analysis that is complementary to other existing spectroscopic methods. Moreover, it is expected to be a useful tool in the study of biomaterials in biological samples.

The liver is the largest internal organ and plays an important part in maintaining the homeostatic balance in the body. It is also one of the primary sites of nanoparticle accumulation. HepG2 spheroids were used as an in vitro model to assess iron oxide (magnetite, Fe3O4 and maghemite, γ-Fe2O3) nanoparticle (NP) toxicity. The development of HepG2 spheroids by the hanging drop method was illustrated through cell viability and tissue morphology assessment of spheroids generated from 1000, 5000, and 10,000 cells per drop. The spheroids generated from 5000 cells per drop had the most optimal configuration. Liver function was assessed by albumin, urea and aspartate transaminase which were expressed in physiologically relevant concentrations by the HepG2 spheroid.Synchrotron X-ray fluorescence (SXRF) was used to assess NP distribution and metal homeostasis. The SXRF studies showed a dose-dependent accumulation of NPs inHepG2 spheroid transverse tissue sections. This accumulation also affected ironhomeostasis by significantly increasing intracellular copper levels. The elemental distribution of calcium and selenium also increased upon challenge with NPs, whichmay be involved in the removal of reactive oxygen species (ROS). Glucose metabolism shifted towards anaerobic respiration in presence NPs, exhibiting the Warburg effect. Transmission electron microscopy also shows intracellular localisation of the iron oxide NPs. Cytokinesis block micronucleus (CBMN) and single-cell gel electrophoresis (comet) assays were used to assess genotoxicity. Both NPs induced a significant increase in micronuclei frequency. Pan-centromeric staining further indicated that the primary mode of action of DNA damage caused by iron oxide NPs was clastogenic. Comet scores showed no significant increases in tail intensities percentages. In conclusion, the HepG2 spheroid model is representative of an in vivo liver due to its ability to mimic glucose metabolism, liver functionality and metal homeostasis responses. Iron oxide NPs cause the HepG2 spheroids to respire anaerobically and disrupt iron homeostasis which can potentially lead to the generation of ROS resulting in the DNA damage., Le foie est le plus grand organe interne et joue un rôle important dans le maintien de l'équilibre homéostatique dans le corps. C'est également l'un des principaux sites d'accumulation de nanoparticules. Les sphéroïdes HepG2 ont été utilisés comme modèle in vitro pour évaluer la toxicité des nanoparticules (NP) d'oxyde de fer (magnétite, Fe3O4 et maghémite, γ-Fe2O3). Le développement des sphéroïdes HepG2 par la méthode des gouttes suspendues a été quantifié par l’évaluation de la viabilité cellulaire et de la morphologie des tissus des sphéroïdes générés à partir de 1000, 5000 et 10 000 cellules par goutte. Les sphéroïdes générés à partir de 5000 cellules par goutte avaient la configuration la plus optimale. La fonction hépatique a été évaluée par l'albumine, l'urée et l'aspartate transaminase, qui ont été exprimées à des concentrations physiologiquement pertinentes par le sphéroïde HepG2.La fluorescence des rayons X synchrotron (SXRF) a été utilisée pour évaluer la distribution des NP et l'homéostasie des métaux. Les études SXRF montrent une accumulation dépendante de la dose de NP dans les coupes transversales de tissu sphéroïde HepG2. Cette accumulation a également affecté l'homéostasie du fer en augmentant de manière significative les niveaux de cuivre intracellulaire. Lesconcentrations du calcium et du sélénium ont également augmenté en présence desNP, ces deux éléments pouvant être impliquées dans l'élimination des espèces réactives de l'oxygène (ROS). Le métabolisme du glucose s'est déplacé vers la respiration anaérobie en présence de NP, montrant l'effet Warburg. La microscopie électronique à transmission montre également la localisation intracellulaire des NP d'oxyde de fer. L'analyse des micronoyaux par blocage de la cytokinèse (CBMN) et l'électrophorèse en gel unicellulaire (comet) ont été utilisées pour évaluer la génotoxicité. Les deux NP ont induit une augmentation significative de la fréquence des micronoyaux. La coloration pan-centromérique a en outre indiqué que le principal mode d'action des dommages à l'ADN causés par les NP d'oxyde de fer était le clastogène. Les résultats comet n'ont montré aucune augmentation significative des pourcentages d'intensité de la queue.En conclusion, le modèle sphéroïde HepG2 est représentatif d'un foie in vivo en raison de sa capacité à imiter le métabolisme du glucose, la fonctionnalité du foie et les réponses à l'homéostasie des métaux. Les NP d'oxyde de fer amènent un métabolismedes sphéroïdes HepG2 anaérobie et perturbent l'homéostasie du fer, ce qui peut potentiellement générer la génération de ROS entraînant des lésions de l'ADN.

Osteoarticular diseases are one of the most common causes of chronic pain, but their diagnosis and understanding can be complex. Different imaging methods are available to describe joint conditions such as radiography, MRI, CT scan or ultrasound, but none of them can depict all the anatomical components in a single image. For the past two decades, X-ray Phase Contrast Imaging (PCI) are constantly being under development because of their superiority for imaging low density objects and its ability to a simultaneous visualization of all tissues. But the extraction of the phase signal is not directly possible, making this information not very often retrieved. The required gold standard instrumentation for PCI is currently a synchrotron facility, limiting clinical applications due to limited access.Several PCI techniques have already been developed and, despite the advances in the literature, they still face many challenges, both instrumental and dosimetric. In this context, the main motivation of this PhD was to propose and develop a phase contrast imaging technique that is easily transferable to conventional sources. The speckle based PCI technique was chosen because, compared to other PCI techniques, it appears to be the most suitable technique for the dose aspects. In addition, it does not require neither an expensive instrumentation nor the specific properties of synchrotron radiation to be implemented. Despite these benefits, it required many algorithmic developments and optimization of the experimental configurations before a transfer on conventional sources.In the first part of the thesis, we propose to show the diagnostic potential of the PCI for osteoarticular applications by imaging anatomical pieces and small animal osteoarticular models. The development and the optimization of speckle based PCI was first performed using the ideal conditions of the European synchrotron source. In particular, a new phase retrieval algorithm has been implemented permitting to reduce the delivered dose while maintaining a constant image quality compared to other speckle based PCI techniques. Several acquisition schemes and experimental conditions were tested. A new instrumental solution for speckle generation has been created. Finally, under these optimized conditions, measurements were made with various conventional X-ray sources. The results obtained have demonstrated the feasibility of the transfer on standard sources under compatible experimental conditions of clinical and pre-clinical imaging routines. This transfer could improve the understanding of the osteoarticular diseases as well as the follow-up of different therapeutic strategies and finally an earlier diagnosis., Les pathologies ostéo-articulaires représentent une des causes les plus fréquentes de douleurs chroniques mais leur diagnostic et leur compréhension peuvent être complexe. Différentes méthodes d'imagerie sont disponibles pour décrire l'état des articulations telles que la radiographie, l'IRM, le scanner ou encore les ultrasons, mais aucune ne permet de visualiser tous les composants anatomiques sur une seule image. Depuis deux décennies, les méthodes d'Imagerie en Contraste de Phase (ICP), dans le domaine des rayons X, font l'objet de développements constants en raison de leur supériorité pour l'imagerie d'objets de faible densité et la visualisation simultanée de tous les tissus. Or l'extraction du signal de phase n'est pas directement mesurable, rendant cette information bien souvent inexploitée. L'instrumentation de référence nécessaire à l'ICP, de par ses propriétés, est à l'heure actuelle une installation synchrotron, limitant les applications cliniques due à l’accès limité à ces instruments.Plusieurs techniques d’ICP ont déjà été développées et, malgré les progrès réalisés dans la littérature, elles doivent encore faire face à de nombreux défis, concernant les aspects instrumentaux et dosimétriques, avant que l’ICP ne devienne une technique non invasive de routine clinique. Dans ce contexte, la principale motivation de cette thèse a été de proposer et développer une technique d’imagerie en contraste de phase qui soit aisément transférable sur source conventionnelle. La technique d’ICP par tavelures a été choisie pour envisager un tel transfert car, par comparaison avec d’autres techniques d’ICP, elle apparaît comme la plus à même de respecter les contraintes dosimétriques. De plus, elle ne nécessite ni une instrumentation coûteuse ni les propriétés spécifiques du rayonnement synchrotron pour être mise en œuvre. En contrepartie de ces avantages, elle demande de nombreux développements algorithmiques et une optimisation des configurations expérimentales avant qu’un déploiement sur sources conventionnelles puisse être envisagé.Dans une première partie de la thèse, nous proposons de montrer le potentiel diagnostic de l’ICP pour les applications ostéo-articulaires en imageant des pièces anatomiques et modèles ostéo-articulaires chez le petit animal. Avant le transfert sur des sources conventionnelles, le développement et l’optimisation de la technique d’ICP par tavelures ont été réalisés dans les conditions idéales du synchrotron européen. En particulier, un nouvel algorithme d’extraction du signal de phase a été implémenté et a permis de réduire la dose délivrée tout en maintenant un rapport signal-sur-bruit constant comparée aux autres techniques d’ICP par tavelures. Plusieurs schémas d’acquisition et conditions expérimentales ont été testés. Une nouvelle solution instrumentale pour générer les tavelures a été créée. Finalement, dans ces conditions optimisées, des mesures ont été réalisées auprès de différentes sources de rayons X conventionnelles. Les résultats obtenus ont ainsi pu démontrer la faisabilité de ce transfert de l’ICP par tavelures sur des sources standards dans des conditions expérimentales compatibles des routines d’imagerie clinique et pré-clinique. Ce transfert pourrait améliorer la compréhension des maladies, le suivi des différentes stratégies thérapeutiques et le diagnostic précoce dans le cas des maladies ostéo-articulaires comme dans le cas d’autres pathologies.

In China, a severe musculoskeletal disease called Kashin-Beck disease (KBD) is largely endemic over a large geographical area. It has been reported that more than 2.5 million people in China suffer from KBD and about 30 million people are at risk. Geological and epidemiological investigations have shown that a strong correlation exists between the location of selenium (Se) deficient soils and the distribution of KBD in the population. The disease is manifested as degradation of the matrix, cell necrosis mainly in the articular and growth plate cartilage, which can result in growth retardation, secondary osteoarthrosis, and disability in daily life. The worst forms of this disease tend to start in childhood, which may lead to dwarfism. Selenium is present everywhere in the environment (water, air, soils) and it is mainly incorporated to the human organism through the daily diet (water, cereals). Although this trace nutriment element is essential for normal cellular function. Most of the selenium-related -functions and pathways remain incompletely understood. Whilst vital for normal function, it is toxic at concentration slightly higher than that required by the body. Consequently, it is present within the organism in parts per billion (microgram per liter) making it difficult to localize and analyze its role in metabolism. Despite being a trace element it is an essential component of antioxidant and anti-inflammatory-related proteins that protect cells against oxidative attack. Furthermore, several studies have exposed the role selenium plays in tissue development such as in articular cartilage. This action seems to be mediated via selenoproteins that are indirectly involved in normal cartilage growth and homeostasis. In the USA, a clinical study has shown strong evidence that Se-deficiency influences cartilage metabolism inducing a favorable environment for the onset and the progression of osteoarthritis. Even if the selenium is not the only factor in the development of degenerative joint disease, it is highly likely that its absence impacts its growth and development of articular cartilage. The main focus of this study was then to understand better the role of Se in the normal metabolic processes of articular cartilage. Cultures of articular cartilage explants were used on a previously validated in vitro model of tissue maturation to analyze the role of selenium in growth and development. Physical and chemical experiments were preformed to understand how the presence of selenium affects tissue organization. It has been possible to determine a fundamental recurrent pattern of Se-distribution in the tissue. It appears to be localized at cell-matrix interfaces and it can be hypothesized that Se plays role in cell signaling or mechanotransduction. Biomechanical, structural and molecular analyses have been made to characterize the extracellular matrix of articular cartilage treated with different concentrations of Se-level. We discovered that Se-deficiency induces morphological changes in the cartilage matrix during the fast maturation-like process, which could be related to degenerative-like morphology of the cartilage. This could potentially be associated with degenerative changes that occur in KBD patient during childhood. This project is a prospective work for a potential future enhancement of the regenerative or preventive treatments for specific musculoskeletal diseases with a metabolic component., En Chine, une grave maladie musculo-squelettique appelée la maladie de Kashin-Beck (KBD) se retrouve distribué sur une large zone géographique. Cette maladie touche plus de deux millions d'individus, notamment dans le centre de la Chine, et il est admis que plus de 30 millions d’individus seraient à risque. Des études géologiques et épidémiologiques ont montré une forte corrélation entre les zones de déficience en Se dans les sols et de KBD. KBD est une ostéoarthropathie, caractérisée par la destruction des chondrocytes du cartilage, très douloureuse et invalidantes, pouvant conduire à des déformations articulaires importantes. Le sélénium (Se) est présent partout dans l'environnement (eau, air, sols) et nos besoins physiologiques en Se sont couverts par notre alimentation quotidienne (eau, céréales). Bien que cet élément trace soit un nutriment essentiel pour la fonction cellulaire normale, ses mécanismes d’action ainsi que les transformations métaboliques de ses composés dans le corps humain ne sont toujours pas bien déterminés. Toutefois, à une dose un peu supérieure à la dose recommandée, il peut, selon la forme chimique ingéré, devenir toxique. Par conséquent, on retrouve le Se en très faible quantité (µg/L) dans l'organisme, ce qui rend difficile sa localisation et l’analyse de son rôle dans le métabolisme. Le Se fait partie de sites biologiquement actifs de protéines impliquées dans les mécanismes antioxydants de défense et le contrôle rédox des réactions intracellulaires. En outre, plusieurs études ont mis en évidence le rôle que joue de Se dans le développement des tissus tels que le cartilage articulaire. Cette action semble être médiée par l'intermédiaire de sélénoprotéines et seraient indirectement impliqués dans la croissance du cartilage normal et l'homéostasie. Aux Etats-Unis, une étude clinique a montré des preuves solides de l’influences d’un déficit en Se dans le métabolisme du cartilage conduisant un environnement favorable à l'apparition et la progression de l'arthrose. Même si le Se n’est pas le seul facteur dans le développement de maladies, il est fort probable que son absence impacte la croissance et le développement du cartilage articulaire. Un modèle in vitro de maturation accélérée du cartilage articulaire (explants) nous a permis d’analyser l'impact du sélénium dans la croissance et le développement de ce tissu. Des expériences biologiques, biophysiques et chimiques ont été réalisées pour comprendre comment la présence de Se affecte l'organisation des tissus. Un schéma récurrent de la distribution du Se dans le tissu a été découvert. Il semble être localisé au niveau des interfaces cellule-matrice, offrant des hypothèses intéressantes pour de futures études sur le rôle potentiel du Se dans la signalisation cellulaire ou transduction mécanique. Des analyses biomécaniques, structurelles et moléculaires ont été faites pour caractériser la matrice extracellulaire du cartilage articulaire traités avec différentes concentrations de Se. Il semble être localisé au niveau des interfaces cellule-matrice, ce qui suggère que le Se joue un rôle dans la signalisation cellulaire ou transduction mécanique. Des analyses biomécaniques, structurelles et moléculaires ont été faites pour caractériser la matrice du cartilage articulaire traités avec différentes concentrations de Se. Nous avons découvert qu’un déficit en Se peut induire à une morphologie proche de celle de l'arthrose lors de la maturation du cartilage immature. Cependant, le rôle exact de ce déficit en Se induisant ce type de phénotype reste inconnu. Ce projet contribue à une meilleure compréhension du Se dans le cartilage tout en montrant les difficultés d’étude du Se dans les milieux biologiques et les techniques permettant d’y répondre, mais aussi souligne l’importance de prendre en compte le Se comme élément important de traitements régénérateurs ou préventifs pour ce types de maladies.

This work presents some recent developments in the field of hard X-ray imaging appliedto biomedical research. As the discipline is evolving quickly, new questions appear andthe list of needs becomes bigger. Some of them are dealt with in this manuscript.It has been shown that the ID22NI beamline of the ESRF can serve as a proper experimentalsetup to investigate diverse aspects of cellular research. Together with its highspatial resolution, high flux and high energy range the experimental setup providesbigger field of view, is less sensitive to radiation damages (while taking phase contrastimages) and suits well chemical analysis with emphasis on endegeneous metals (Zn, Fe,Mn) but also with a possibility for for exogoneous one’s like these found in nanoparticles(Au, Pt, Ag) study.Two synchrotron-based imaging techniques, fluorescence and phase contrast imagingwere used in this research project. They were correlated with each other on a numberof biological cases, from bacteria E.coli to various cells (HEK 293, PC12, MRC5VA,red blood cells).The explorations made in the chapter 5 allowed preparation of more establishedand detailed analysis, described in the next chapter where both techniques, X-ray fluorescenceand phase contrast imaging, were exploited in order to access absolute metalprojected mass fraction in a whole cell. The final image presents for the first timetrue quantitative information at the sub-cellular level, not biased by the cell thickness.Thus for the first time a fluorescence map serves as a complete quantitative image of acell without any risk of misinterpretation. Once both maps are divided by each otherpixel by pixel (fluorescence map divided by the phase map) they present a completeand final result of the metal (Zn in this work) projected mass fraction in ppm of dryweight. For the purpose of this calculation the analysis was extended to calibration(non-biological) samples. Polystyrene spheres of a known diameter and known densityworked very well here and allowed validation of the presented method. Different images(phase map, AFM, STIM) and profiles were compared and statement on the high accuracyof phase contrast imaging for the thickness/structures determination was made.The result on true metal projected mass fraction represents a first step to an absolutesub-cellular analysis and certainly can be improved to even closer reflect on reality.All the measurements were taken on freeze-dried cells. Thus the result is in ppm ofdry weight. In fact the measurement would have even deeper meaning if it was madeon hydrated cells. For the moment this is not possible with the existing setup of theID22NI beamline but will be possible in the future with a new beamline devoted tonano science - NINA (Nano-Imaging and Nano-Analysis). The new beamline will befurnished with a cryostage and X-ray imaging will be made on frozen-hydrated samples.Nevertheless the analysis presented in this manuscript is of undeniable importance toboth the biomedical community and to the ESRF team engaged in the NINA development.To answer the problems of cell irradiation both imaging techniques were exploitedagain. Repeating the phase contrast imaging after the fluorescence scanning allowedto show the changes induced by radiation damage during X-ray fluorescence scan. Thechanges were not only clearly visible but could be as well quantified. Together with thenumerical evaluation of damages, the dose delivered to a cell during the experiment was calculated as well. To complete the picture, a different non synchrotron-basedimaging technique, STIM, was used and compared. It is the first time that phase contrastimaging is used to monitor radiation damage effects during X-ray fluorescencemicroscopy experiments.; Ce travail de thèse présente une combinaison unique d'imagerie X par contraste de phase avec la fluorescence X pour des échantillons biologiques étudiés par nanosonde par fluorescence X excitée par le rayonnement synchrotron. Les récents développements dans ce domaine ouvrent la possibilité d'une imagerie chimique quantitative à l'échelle sub-cellulaire. Ceci a été rendu possible par l'utilisation d'un outil unique qui est la station de nanoimagerie X ID22NI de l'ESRF qui permet de délivrer un faisceau sub-100 nm avec un très haut flux à haute énergie entrainant une sensibilité très haute, de l'ordre de quelques centaines d'atomes pour différents éléments (Fe, Cu, Zn…). Le couplage des informations issues de l'imagerie X par contraste de phase (masse surfacique de la cellule) et de la fluorescence X (masse surfacique des éléments chimiques) a pu être obtenu pour la première fois donnant accès à une cartographie des éléments chimiques constituant les cellules et de leurs fractions massiques absolues associées. Dans l'immédiat, il n'a été possible d'étudier des cellules qui ont été congelées rapidement puis lyophilisées, cependant, une nouvelle ligne de nanoimagerie, NINA, en construction à l'ESRF, fonctionnera comme un cryomicroscope et permettra l'analyse 2D/3D d'échantillons biologiques ou non congelés hydratés. L'extension de l'imagerie chimique 2D présentée dans ce travail à une imagerie 3D représente une importante avancée pour bon nombre de problématiques scientifiques en biologie. Une des limitations de ce type d'analyse est celle des dommages radio-induits à la suite de l'irradiation de l'échantillon par un haut flux de particules ionisantes. Il existe que peu ou pas d'étude sur les effets de la nanoanalyse par fluorescence X sur les cellules lyophilisées. Nous avons combiné l'imagerie de phase à l'imagerie par fluorescence X ce qui nous permis de conclure à une rétractation des structures cellulaires accompagnée d'une volatilisation des éléments du fait de l'irradiation lors de l'analyse par fluorescence X. Ces aspects ont été confortés par des analyses utilisant une technique complémentaire non-synchrotron de microscopie ionique en transmission et à balayage (STIM). Plus important encore, nous apportons ainsi un outil rapide et non-destructif pour la cellule (imagerie X de phase) qui permet de corriger la perte de masse due à la volatilisation d'éléments légers (C, H, O, N) de la matrice cellulaire. Cette démarche permet de fiabiliser l'analyse quantitative de la composition chimique cellulaire. Cette approche sera précieuse pour corriger ces effets de perte de masse lors de futures analyses tomographiques de cellules entières congelées hydratées. Nous avons également contribué à l'étude de distribution intracellulaire de nouvelles nanoparticules d'or ou de platine fonctionnalisées. Nous avons pu exploiter les données issues de la fluorescence X pour estimer le nombre de nanoparticules et la taille des clusters internalisés au sein des cellules. Toutefois, des expériences dédiées pour des analyses sur un plus grand nombre de cellules auxquelles l'imagerie X par contraste de phase serait menée en parallèle permettraient surement de préciser plus finement ces aspects quantitatifs sur le nombre de nanoparticules intracellulaires. Dans l'ensemble ce travail ouvre la possibilité d'une imagerie chimique quantitative absolue sub-cellulaire en 2D ou 3D avec la perspective d'imagerie corrélative avec de nombreuses techniques complémentaires notamment la microscopie électronique à transmission pour l'ultrastructure, la microscopie de fluorescence pour la localisation de proteines d'intérêts et d'autres techniques d'analyses chimiques telles le NanoSIMS ou le nano-PIXE.

Manganese (Mn2+) is an essential element for human body. The paramagnetic properties of Mn2+ permit it use as a contrast agent for MRI (Mn-MRI or MEMRI). Analogue of calcium (Ca2+), it enters neurons primarily by calcium channels. It is then transported along microtubules to the synapse where it is released and then captured by other neurons. Thus, it can account for the anterograde and retrograde axonal transport. The MEMRI approach can provide unique information about cerebral functional connectivity. Two problems limit the use of this powerful tool for in vivo imaging: (i) At high doses, Mn2+ is toxic to the body and can cause serious problem of the central nervous system, called manganism. The level and the mechanisms of toxicity are poorly understood. (ii) The mode of manganese transport in the MEMRI approach is unclear. To address these two issues, we undertook a study coupling MRI and synchrotron microscopy to study the Mn 2+ behavior in vivo. We characterized the cellular and subcellular distributions of Mn and other metals in "pseudo neurons" cell line N2A, primary cultures of hippocampal neurons, andin hippocampal slices from rats. In parallel, we studied the effects of Mn on brain metabolism by proton-HRMAS NMR . In parallel, weevaluated MEMRI in MAP6 KO mice which exhibit a deficit in microtubule stabilizing protein, to assess the functional connectivity of the thalamocortical tract. Key words hippocampus, MAP6, manganese, metabolism, metal, neuron, MRI, rodent, synchrotron., Résumé Le manganèse (Mn2+) est un élément essentiel du corps humain. Ses propriétés paramagnétiques permettent son utilisation comme agent de contraste pour l'IRM (Mn-MRI ou MEMRI). Analogue du calcium (Ca2+), il pénètre les neurones essentiellement par les canaux calciques. Il est ensuite transporté le long des microtubules jusqu'aux synapses où il est libéré, puis capturé par les autres neurones. Ainsi, il peut rendre compte du transport axonal antérograde et rétrograde. L'approche MEMRI peut ainsi apporter des informations uniques sur la connectivité fonctionnelle cérébrale. Toutefois, deux problèmes limitent l'emploi de ce puissant outil d'imagerie in vivo : (i) A doses élevées, le Mn2+ est toxique pour l'organisme et peut provoquer une atteinte grave du système nerveux central, appelé manganisme. Les niveaux et les mécanismes de toxicité sont mal connus. (ii) Le mode de transport du manganèse dans l'approche MEMRI est mal connu. Afin d'apporter des éléments de réponse à ces deux problèmes, nous avons entrepris une étude couplant IRM et microscopie synchrotron pour mieux comprendre le comportement du Mn2+ in vivo. Nous avons précisé les distributions cellulaire et sub-cellulaire du Mn et d'autres métaux pour un modèle de cellules de type neuronal (lignée de neuroblastome N2A), pour des cultures primaires de neurones hippocampiques, mais aussi au niveau de coupes d'hippocampe de rats. En parallèle, nous avons étudié les effets du Mn sur le métabolisme cérébral par une technique de RMN-HRMAS du proton. Pour compléter ce travail, nous avons mis en œuvre l'imagerie MEMRI chez les souris KO MAP6 présentant un déficit d'une protéine stabilisatrice des microtubules pour évaluer la connectivité fonctionnelle du tract thalamo-cortical. Mots clés Hippocampe, MAP6, manganèse, métabolisme, métal, neurone, MRI, rongeurs, synchrotron.

Manganese (Mn2+) is an essential element for human body. The paramagnetic properties of Mn2+ permit it use as a contrast agent for MRI (Mn-MRI or MEMRI). Analogue of calcium (Ca2+), it enters neurons primarily by calcium channels. It is then transported along microtubules to the synapse where it is released and then captured by other neurons. Thus, it can account for the anterograde and retrograde axonal transport. The MEMRI approach can provide unique information about cerebral functional connectivity. Two problems limit the use of this powerful tool for in vivo imaging: (i) At high doses, Mn2+ is toxic to the body and can cause serious problem of the central nervous system, called manganism. The level and the mechanisms of toxicity are poorly understood. (ii) The mode of manganese transport in the MEMRI approach is unclear. To address these two issues, we undertook a study coupling MRI and synchrotron microscopy to study the Mn 2+ behavior in vivo. We characterized the cellular and subcellular distributions of Mn and other metals in "pseudo neurons" cell line N2A, primary cultures of hippocampal neurons, andin hippocampal slices from rats. In parallel, we studied the effects of Mn on brain metabolism by proton-HRMAS NMR . In parallel, weevaluated MEMRI in MAP6 KO mice which exhibit a deficit in microtubule stabilizing protein, to assess the functional connectivity of the thalamocortical tract. Key words hippocampus, MAP6, manganese, metabolism, metal, neuron, MRI, rodent, synchrotron., Résumé Le manganèse (Mn2+) est un élément essentiel du corps humain. Ses propriétés paramagnétiques permettent son utilisation comme agent de contraste pour l'IRM (Mn-MRI ou MEMRI). Analogue du calcium (Ca2+), il pénètre les neurones essentiellement par les canaux calciques. Il est ensuite transporté le long des microtubules jusqu'aux synapses où il est libéré, puis capturé par les autres neurones. Ainsi, il peut rendre compte du transport axonal antérograde et rétrograde. L'approche MEMRI peut ainsi apporter des informations uniques sur la connectivité fonctionnelle cérébrale. Toutefois, deux problèmes limitent l'emploi de ce puissant outil d'imagerie in vivo : (i) A doses élevées, le Mn2+ est toxique pour l'organisme et peut provoquer une atteinte grave du système nerveux central, appelé manganisme. Les niveaux et les mécanismes de toxicité sont mal connus. (ii) Le mode de transport du manganèse dans l'approche MEMRI est mal connu. Afin d'apporter des éléments de réponse à ces deux problèmes, nous avons entrepris une étude couplant IRM et microscopie synchrotron pour mieux comprendre le comportement du Mn2+ in vivo. Nous avons précisé les distributions cellulaire et sub-cellulaire du Mn et d'autres métaux pour un modèle de cellules de type neuronal (lignée de neuroblastome N2A), pour des cultures primaires de neurones hippocampiques, mais aussi au niveau de coupes d'hippocampe de rats. En parallèle, nous avons étudié les effets du Mn sur le métabolisme cérébral par une technique de RMN-HRMAS du proton. Pour compléter ce travail, nous avons mis en œuvre l'imagerie MEMRI chez les souris KO MAP6 présentant un déficit d'une protéine stabilisatrice des microtubules pour évaluer la connectivité fonctionnelle du tract thalamo-cortical. Mots clés Hippocampe, MAP6, manganèse, métabolisme, métal, neurone, MRI, rongeurs, synchrotron.