Combined use of X-ray Fluorescence Microscopy, X-ray Phase Contrast Imaging, Atomic Force Microscopy and X-ray Nanotomography for high resolution quantitative Fe mapping in inflamed cells.

Abstract

The PhD project is based on the applications of several x-ray microscopy techniques for compositional and morphological studies at nanoscale spatial resolution to a biological problem, i.e. the quantitative determination of morphological and compositional properties of epithelial cells. Three x-ray microscopy techniques were exploited in this work: X-ray fluorescence microscopy, X-ray phase contrast imaging and nanotomography, which were made at the ID16NI beamline of the European Synchrotron Radiation Facility in Grenoble, France. In addition to synchrotron-based techniques, also Atomic Force Microscopy was performed. The latter was used for morphology characterization, and forcalibration and comparison purpose. The main aim of this study was to quantitatively determine the map of iron concentration at nanoscale spatial resolution of epithelial cells infected by bacterial pathogens in the presence or absence of lactoferrin (Lf), an iron-chelating glycoprotein of natural immunity. Two experiments have been carried out at ESRF, one on freeze dried cells, and one on frozen hydrated cells this last using the cryo stage foreseen in the Id16 NI beamline, in order to examine cells as close as possible to their native state, and to avoid radiation damage. The measurement and data analysis protocols have been carefully studied for optimal combination of all the techniques, to give quantitative results. Iron concentration and mass fraction maps have been obtained, which give an insight about the modification of iron spatial distribution under the influence of lactoferrin. Moreover, for the first time it has been demonstrated the possibility to obtain quantitative element concentration in cells through the combination of x-ray nanotomography in phase contrast and x-ray fluorescence microscopy.