[en] This work presents some recent developments in the field of hard X-ray imaging applied to biomedical research. As the discipline is evolving quickly, new questions appear and the 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 experimental setup to investigate diverse aspects of cellular research. Together with its high spatial resolution, high flux and high energy range the experimental setup provides bigger field of view, is less sensitive to radiation damages (while taking phase contrast images) and suits well chemical analysis with emphasis on endogenous metals (Zn, Fe, Mn) but also with a possibility for exogenous one's like these found in nanoparticles (Au, Pt, Ag) study. Two synchrotron-based imaging techniques, fluorescence and phase contrast imaging were used in this research project. They were correlated with each other on a number of 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 established and detailed analysis, described in the next chapter where both techniques, X-ray fluorescence and phase contrast imaging, were exploited in order to access absolute metal projected mass fraction in a whole cell. The final image presents for the first time true 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 a cell without any risk of misinterpretation. Once both maps are divided by each other pixel by pixel (fluorescence map divided by the phase map) they present a complete and final result of the metal (Zn in this work) projected mass fraction in ppm of dry weight. For the purpose of this calculation the analysis was extended to calibration (non-biological) samples. Polystyrene spheres of a known diameter and known density worked 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 accuracy of phase contrast imaging for the thickness/structures determination was made. The result on true metal projected mass fraction represents a first step to an absolute sub-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 of dry weight. In fact the measurement would have even deeper meaning if it was made on hydrated cells. For the moment this is not possible with the existing setup of the ID22NI beamline but will be possible in the future with a new beamline devoted to nano science - NINA (Nano-Imaging and Nano-Analysis). The new beamline will be furnished with a cryo-stage and X-ray imaging will be made on frozen-hydrated samples. Nevertheless the analysis presented in this manuscript is of undeniable importance to both the biomedical community and to the ESRF team engaged in the NINA development. To answer the problems of cell irradiation both imaging techniques were exploited again. Repeating the phase contrast imaging after the fluorescence scanning allowed to show the changes induced by radiation damage during X-ray fluorescence scan. The changes were not only clearly visible but could be as well quantified. Together with the numerical evaluation of damages, the dose delivered to a cell during the experiment was calculated as well. To complete the picture, a different non synchrotron-based imaging technique, STIM, was used and compared. It is the first time that phase contrast imaging is used to monitor radiation damage effects during X-ray fluorescence microscopy experiments. (author)

[en] 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

[en] Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to study radiation effects on cells. Experiments were performed on freeze-dried cells at the nano-imaging station ID22NI of the European synchrotron radiation facility. Quantitative phase contrast imaging provides maps of the projected mass and is used to evaluate the structural changes due to irradiation during X-ray fluorescence experiments. Complementary to phase contrast imaging, scanning transmission ion microscopy is performed and doses of all the experiments are compared. We demonstrate the sensitivity of the proposed approach to study radiation-induced damage at the sub-cellular level.