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.
