Phase-contrast X-ray lung imaging has broken new ground in preclinical respiratory research by improving contrast at air/tissue interfaces. To minimize blur from respiratory motion, intubation and mechanical ventilation is commonly employed for end-inspiration gated imaging at synchrotrons and in the laboratory. Inevitably, the prospect of ventilation induced lung injury (VILI) renders mechanical ventilation a confounding factor in respiratory studies of animal models. Here we demonstrate proof-of-principle 3D imaging of the tracheobronchial tree in free-breathing mice without mechanical ventilation at radiation levels compatible with longitudinal studies. We use a prospective gating approach for end-expiration propagation-based phase-contrast X-ray imaging where the natural breathing of the mouse dictates the acquisition flow. We achieve intrapulmonary spatial resolution in the 30-μm-range, sufficient for resolving terminal bronchioles in the 60-μm-range distinguished from the surrounding lung parenchyma. These results should enable non-invasive longitudinal studies of native state murine airways for translational lung disease research in the laboratory.

X-ray phase-contrast tomography (XPCT) is a non-destructive, threedimensional imaging modality that provides higher contrast in soft tissue than absorption-based CT and allows one to cover the cytoarchitecture from the centi-and millimeter scale down to the nanoscale. To further increase contrast and resolution of XPCT, for example, in view of addressing connectivity issues in the central nervous system (CNS), metal staining is indispensable. However, currently used protocols, for example, based on osmium and/or uranium are less suited for XPCT, due to an excessive β∕δ-ratio. In this work, we explore the suitability of different staining agents for XPCT. Particularly, neodymium(III)-acetate (NdAc), which has recently been proposed as a non-toxic, non-radioactive easy to use alternative contrast agent for uranyl acetate (UAc) in electron microscopy, is investigated. Due to its vertical proximity to UAc in the periodic table, similar chemical but better suited optical properties for phase contrast can be expected.Approach: Differently stained whole eye samples of wild type mouse and tissues of the CNS are embedded into EPON epoxy resin and scanned using synchrotron as well as with laboratory radiation. Phase retrieval is performed on the projection images, followed by tomographic reconstruction, which enables a quantitative analysis based on the reconstructed electron densities. Segmentation techniques and rendering software is used to visualize structures of interest in the sample. Results:We show that staining neuronal samples with NdAc enhances contrast, in particular for laboratory scans, allowing high-resolution imaging of biological soft tissue in-house. For the example of murine retina, specifically rods and cones as well as the sclera and the Ganglion cell layer seem to be targeted by the stain. A comparison of electron density by the evaluation of histograms allowed to determine quantitative measures to describe the difference between the examined stains. Conclusion:The results suggest NdAc to be an effective stain for XPCT, with a preferential binding to anionic groups, such as phosphate and carboxyl groups at cell surfaces, targeting certain layers of the retina with a stronger selectivity compared to other staining agents. Due to the advantageous X-ray optical properties, the stain seems particularly well-suited for phase contrast, with a comparably small number density and an overall superior image quality at laboratory sources.

As the central organ of the respiratory system, the human lung is responsible for supplying oxygen to the blood, which reaches the erythrocytes by diffusion through the alveolar walls and is then distributed throughout the body. By exploiting the difference in electron density detected by a phase shift in soft tissue, high-resolution X-ray phase-contrast computed tomography (XPCT) can resolve biological structures in a sub-μm range, shedding new light on the three-dimensional structure of the lungs, physiological functions and pathological mechanisms.

This work presents both synchrotron and laboratory XPCT results of postmortem tissue from autopsies and biopsies embedded with various preparation protocols such as precision-cut lung slices, cryogenically fixed lung tissue, as well as paraffin and alcohol fixed tissue. The selection of pathological abnormalities includes channel of Lambert, bronchus-associated lymphoid tissue, alveolar capillary dysplasia with misalignment of pulmonary veins. Subsequently, quantification and visualization approaches are presented.

The overall high image quality even of in-house XPCT scans for the case of FFPE biopsies can be exploited for a wide range of pulmonary pathologies and translated to dedicated and optimized instrumentation which could be operated in clinical setting. By using synchrotron radiation, contrast can be further increased to resolve sub-μm sized features down to the sub-cellular level. The results demonstrate that a wide range of preparation protocols including sample mounting in liquids can be used. 

With XPCT, poorly understood 3D structures can be identified in larger volume overview and subsequently studied in more detail at higher resolution. With the full 3D structure, the respective physiological functions of airways or vascular networks, and the different pathophysiologic mechanisms can be elucidated or at least underpinned with structural data. Moreover, synchrotron data can be used to validate laboratory protocols and provide ground truth for standardizing the method.


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