3D Multimodal X-ray microscopy of biological specimens

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Long penetration depth of X-rays makes them particularly suitable for non-destructive volumetric investigations of whole cells and tissue sections, providing structural and elemental specificity at nanoscale spatial resolutions.X-ray ptychography is a promising imaging technique for sub-100-nm structural studies of weakly-scattering extended biological specimens. It provides quantitative phase-contrast maps revealing the morphology of an investigated object. In addition, its scanning nature allows for a simultaneous acquisition of nanoscale X-ray fluorescence, yielding the element distributions at an unmatched sensitivity.Thus, the goal of this PhD project has been to combine X-ray fluorescence and ptychography to enable a robust correlation of elemental distributions with respect to the cellular morphology. Exploiting a highly intense and coherent X-ray beam at beamline P11 of the low-emittance synchrotron light source PETRA III in Hamburg, Germany, a versatile multimodal scanning X-ray microscope was developed in the framework of this PhD project. This thesis describes the consecutive stages of the microscope’s development and highlights its application in structural determination of biological samples.In the first stage of this PhD project, a 2D long-range scanning unit was developed, enabling seamless, serial measurements of many targets at nanometer precision. With this setup, the PhD work allowed to optimise the correlative imaging method at higher incident photon energies for mapping of first-row transition metals up to iron, while using a nano-focussed beam. In this way, it addressed limitations of the so-far presented demonstrations, restrained to light-element mapping in the context of organelles of single cells. The correlative imaging method was then used to quantify the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers in the context of their sub-cellular structure obtained by ptychography. In the second application, the calcium content in a human bone matrix was mapped in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was then used to remove the mass-thickness effect from the raw calcium map.In the second stage of this work, the concept of concurrent ptychography and X-ray fluorescence was extended to 3D correlative imaging in the framework of computed tomography. For this purpose, an upgraded 3D scanning X-ray microscope was built, commissioned, and used in volumetric imaging of two specimens, using ptychographic X-ray computed tomography. The final performance of the 3D microscope reached far beyond the throughputs of the available ptychographic tomography setups which, until now, have hindered application of the method by a broader biomedical community. A tomogram of the nano-porous glass demonstrated the fastest on-the-fly ptychographic tomography, to date, at an isotropic spatial resolution of 52 nm. In the second application, ptychographic tomography of a chiton’s radular tooth provided a quantitative insight into one of the nature’s hardest biominerals, at a Nyquist-sampling limited spatial resolution of 65 nm.The work has been concluded with a prospect for future operation and opportunities of the correlative method of simultaneous ptychographic and X-ray fluorescence imaging at upcoming diffraction-limited synchrotron light sources