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Even though the validity of x-ray computed tomography in the analysis of biomedical samples is nowadays undisputed, the more recent imaging techniques and more advanced instruments (such as synchrotrons) are still relatively unknown to many medical doctors that could benefit from them.The doctoral work presented in this thesis joins a collective effort from the imaging community to demonstrate potential applications of advanced x-ray and neutron imaging methods to preclinical medical research, with the hope of contributing to reach a “critical mass” in the medical community and in the public opinion as well.Two main lines of work are detailed, one focused on the ex vivo evaluation of corrosion processes of magnesium-based biodegradable implants for osteosynthesis, the other dedicated to the assessment of neuropathy in human gastroenteric dysmotility. The aimed endpoint was to develop pipelines, from image acquisition all the way to data quantification, that could be used by other research groups with similar questions and may inspire future interdisciplinary collaborations between medicine, natural science and engineering.In the first line of work, we have attempted to employ synchrotron-radiation micro-computed tomography (µCT) coupled with in situ loading tests to assess the mechanical properties of the bone-implant interface (Paper I). We have revealed the crucial importance of the radiation dose deposited on the sample, and that the mechanical loading geometry should be accurately determined in the planning steps of the experiment. Moving away from the mechanical testing, we have also explored a novel three-dimensional analysis of the corrosion by-products of biodegradable implants by combining x-ray µCT, neutron µCT and x-ray fluorescence mapping (Papers IV and V). The second line of work has assessed the potential of x-ray phase-contrast µCT and nano-resolution holotomography as ways to perform virtual histology of unstained peripheral and autonomic neural tissue. In full-thickness biopsies of the myenteric nervous system, qualitative and potentially quantitative differences have been shown between controls and patients affected by gastrointestinal dysmotility (Paper II). In unstained skin biopsies, the methods have failed to visualise peripheral nerves, but we could identify structural changes in the connective tissue of some patients when compared to controls and other patients (Paper III).

Even though the validity of x-ray computed tomography in the analysis of biomedical samples is nowadays undisputed, the more recent imaging techniques and more advanced instruments (such as synchrotrons) are still relatively unknown to many medical doctors that could benefit from them.The doctoral work presented in this thesis joins a collective effort from the imaging community to demonstrate potential applications of advanced x-ray and neutron imaging methods to preclinical medical research, with the hope of contributing to reach a “critical mass” in the medical community and in the public opinion as well.Two main lines of work are detailed, one focused on the ex vivo evaluation of corrosion processes of magnesium-based biodegradable implants for osteosynthesis, the other dedicated to the assessment of neuropathy in human gastroenteric dysmotility. The aimed endpoint was to develop pipelines, from image acquisition all the way to data quantification, that could be used by other research groups with similar questions and may inspire future interdisciplinary collaborations between medicine, natural science and engineering.In the first line of work, we have attempted to employ synchrotron-radiation micro-computed tomography (µCT) coupled with in situ loading tests to assess the mechanical properties of the bone-implant interface (Paper I). We have revealed the crucial importance of the radiation dose deposited on the sample, and that the mechanical loading geometry should be accurately determined in the planning steps of the experiment. Moving away from the mechanical testing, we have also explored a novel three-dimensional analysis of the corrosion by-products of biodegradable implants by combining x-ray µCT, neutron µCT and x-ray fluorescence mapping (Papers IV and V). The second line of work has assessed the potential of x-ray phase-contrast µCT and nano-resolution holotomography as ways to perform virtual histology of unstained peripheral and autonomic neural tis

Background Dense and unbiased cellular-resolution representations of extended volumetric central nervous system soft-tissue anatomy are difficult to obtain, even in experimental post-mortem settings. Interestingly, X-ray phase-contrast computed tomography (X-PCI-CT), an emerging soft-tissue-sensitive volumetric imaging technique, can provide multiscale organ- to cellular-level morphological visualizations of neuroanatomical structure. New Method Here, we tested different nervous-tissue fixation procedures, conventionally used for transmission electron microscopy, to better establish X-PCI-CT-specific sample-preparation protocols. Extracted rat spinal medullas were alternatively fixed with a standard paraformaldehyde-only aldehyde-based protocol, or in combination with glutaraldehyde. Some specimens were additionally post-fixed with osmium tetroxide. Multiscale X-PCI-CT datasets were collected at several synchrotron radiation facilities, using state-of-the-art setups with effective image voxel sizes of 3.03 to 0.33 μm3, and compared to high-field magnetic resonance imaging, histology and vascular fluorescence microscopy data. Results Multiscale X-PCI-CT of aldehyde-fixed spinal cord specimens resulted in dense histology-like volumetric representations and quantifications of extended deep spinal micro-vascular networks and of intra-medullary cell populations. Osmium post-fixation increased intra-medullary contrast between white and gray-matter tissues, and enhanced delineation of intra-medullary cellular structure, e.g. axon fibers and motor neuron perikarya. Comparison with Existing Methods Volumetric X-PCI-CT provides complementary contrast and higher spatial resolution compared to 9.4 T MRI. X-PCI-CT’s advantage over planar histology is the volumetric nature of the cellular-level data obtained, using samples much larger than those fit for volumetric vascular fluorescence microscopy. Conclusions Deliberately choosing (post-)fixation protocols tailored for optimal nervous-tissue structural preservation is of paramount importance in achieving effective and targeted neuroimaging via the X-PCI-CT technique.

Phase-Contrast Tomography (PCT) is becoming an important technique for nondestructive, in-situ characterization of soft and hard condensed matter. This thesis sheds light on our progress in developing novel tomographic reconstruction algorithms in combination with in-situ experimental approaches that employ propagation-based PCT. These developments are aimed to improve the spatial and temporal resolutions of in-situ studies of the soft and hard condensed matter. The mechanical properties of materials such as steel are, to a large extent, defined by its microstructure. Propagation-based PCT (i.e. holotomography) can be used to visualize the microstructures associated with small variations in density or composition in the bulk of the specimen. This imaging technique has the advantage of being non-destructive and allows to carry out in-situ, time-resolved observations of miscrostructural changes during dynamic processes such as solid-state phase transformations. Sufficient spatial and temporal resolution can be achieved only when an extremely bright x-ray source is used. In order to test whether a novel table-top x-ray source, the MIRRORCLE-6X, can be used for implementation of a propagation-based PCT system, we have carried out a series of experiments. The characteristics of a propagation-based PCI system that employs the MIRRORCLE-6X were analyzed. In our further research we have focused on the experiments that could be performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. We reported on the investigation of the cementite microstructure in carbon steel. In this work we present the reconstruction of the three-dimensional morphology of cementite grains in the bulk of steel using a non-destructive imaging technique – x-ray Phase-Contrast Tomography (PCT). Complementary information about the crystalline structure of the ferrite grains surrounding the cementite is obtained using x-ray Diffraction-Contrast Tomography (DCT). The work was continued by a time-resolved x-ray PCT investigation of the austenite grain growth during the ferrite-to-austenite phase transformation in low-carbon steel. Experimental data allows to visualize the evolution of the three-dimensional morphology of the austenite-ferrite interfaces with a spatial resolution on the order of 1 ?m and a time resolution of less than 9 minutes. The in-situ measurements show that the theory for grain growth during diffusional solid-state phase transformations needs to be extended in order to describe the drastically lower mobility that we observed for flat interfaces compared to curved interfaces. We observe that some flat interfaces are completely immobile, which is not predicted by the state-of-the-art theories and showed - for the first time - that new computational model for grain growth are needed. Quantitative image reconstruction based on PCT data requires solving two inverse problems: phase retrieval and tomographic reconstruction. In most cases, the problem of phase retrieval is ill-posed and requires that some type of a prior knowledge about the reconstructed image (i.e. particular regularization method) is used. We present a novel algebraic approach suitable for phase retrieval using various linear models. In this approach Total Variation (TV) minimization is used for regularization of the linearized inverse problem by promoting the solution with a sparse gradient magnitude (i.e. piece-wise constant solution). In that case prior knowledge about the reconstructed image may allow to (partially) recover the unknown spatial frequencies that are undefined by the experimental data. The problem of tomographic reconstruction based on PCT data can be solved using a similar approach. We conclude our investigation of the image reconstruction algorithms by introducing several algebraic approaches suitable for tomographic reconstruction based on PCT data. In these approaches TV minimization is used to find a regularized solution to an underdetermined linear system based on a linearized representation of the PCT. When the density of the reconstructed object is piece-wise constant (or close to it) a virtually artifact-free solution can be computed for the tomographic problem. The proposed approach can also be used to radically improve the accuracy of the tomographic reconstruction based on incomplete data (e.g. small number of projections) or data with low signal to noise ratio.

Phase-Contrast Tomography (PCT) is becoming an important technique for nondestructive, in-situ characterization of soft and hard condensed matter. This thesis sheds light on our progress in developing novel tomographic reconstruction algorithms in combination with in-situ experimental approaches that employ propagation-based PCT. These developments are aimed to improve the spatial and temporal resolutions of in-situ studies of the soft and hard condensed matter. The mechanical properties of materials such as steel are, to a large extent, defined by its microstructure. Propagation-based PCT (i.e. holotomography) can be used to visualize the microstructures associated with small variations in density or composition in the bulk of the specimen. This imaging technique has the advantage of being non-destructive and allows to carry out in-situ, time-resolved observations of miscrostructural changes during dynamic processes such as solid-state phase transformations. Sufficient spatial and temporal resolution can be achieved only when an extremely bright x-ray source is used. In order to test whether a novel table-top x-ray source, the MIRRORCLE-6X, can be used for implementation of a propagation-based PCT system, we have carried out a series of experiments. The characteristics of a propagation-based PCI system that employs the MIRRORCLE-6X were analyzed. In our further research we have focused on the experiments that could be performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. We reported on the investigation of the cementite microstructure in carbon steel. In this work we present the reconstruction of the three-dimensional morphology of cementite grains in the bulk of steel using a non-destructive imaging technique – x-ray Phase-Contrast Tomography (PCT). Complementary information about the crystalline structure of the ferrite grains surrounding the cementite is obtained using x-ray Diffraction-Contrast Tomography (DCT). The work was continued by a time-resolved x-ray PCT investigation of the austenite grain growth during the ferrite-to-austenite phase transformation in low-carbon steel. Experimental data allows to visualize the evolution of the three-dimensional morphology of the austenite-ferrite interfaces with a spatial resolution on the order of 1 ?m and a time resolution of less than 9 minutes. The in-situ measurements show that the theory for grain growth during diffusional solid-state phase transformations needs to be extended in order to describe the drastically lower mobility that we observed for flat interfaces compared to curved interfaces. We observe that some flat interfaces are completely immobile, which is not predicted by the state-of-the-art theories and showed - for the first time - that new computational model for grain growth are needed. Quantitative image reconstruction based on PCT data requires solving two inverse problems: phase retrieval and tomographic reconstruction. In most cases, the problem of phase retrieval is ill-posed and requires that some type of a prior knowledge about the reconstructed image (i.e. particular regularization method) is used. We present a novel algebraic approach suitable for phase retrieval using various linear models. In this approach Total Variation (TV) minimization is used for regularization of the linearized inverse problem by promoting the solution with a sparse gradient magnitude (i.e. piece-wise constant solution). In that case prior knowledge about the reconstructed image may allow to (partially) recover the unknown spatial frequencies that are undefined by the experimental data. The problem of tomographic reconstruction based on PCT data can be solved using a similar approach. We conclude our investigation of the image reconstruction algorithms by introducing several algebraic approaches suitable for tomographic reconstruction based on PCT data. In these approaches TV minimization is used to find a regularized solution to an underdetermined linear system based on a linearized representation of the PCT. When the density of the reconstructed object is piece-wise constant (or close to it) a virtually artifact-free solution can be computed for the tomographic problem. The proposed approach can also be used to radically improve the accuracy of the tomographic reconstruction based on incomplete data (e.g. small number of projections) or data with low signal to noise ratio.