Recent advancements in propagation-based phase-contrast imaging, such as hierarchical imaging, have enabled the visualization of internal structures in large biological specimens and material samples. However, wavefront marker-based techniques, which provide quantitative electron density information, face challenges when imaging larger objects due to stringent beam stability requirements and potential structural changes in objects during longer measurements. Extending the fields-of-view of these methods is crucial for obtaining comparable quantitative results across beamlines and adapting to the smaller beam profiles of fourth-generation synchrotron sources. We introduce a novel technique combining an adapted eigenflat optimization with deformable image registration to address the challenges and enable quantitative high-resolution scans of centimeter-sized objects with micrometre resolution. We demonstrate the potential of the method by obtaining an electron density map of a rat brain sample 15 mm in diameter using speckle-based imaging, despite the limited horizontal field-of-view of 6 mm of the beamline (PETRA III, P05, operated by Hereon at DESY). This showcases the ability of the technique to significantly widen the range of application of wavefront marker-based techniques in both biological and materials science research.
In histology, the widely used staining agents are hematoxylin and eosin (H&E), with hematoxylin marking cell nuclei and eosin staining the cytoplasm. By this, the color-coded information enables the early identification of histopathological changes using optical microscopy. However, the traditional histological process has significant drawbacks: the irreversible nature of tissue preparation often results in sample damage during dehydration, embedding, and sectioning, which can lead to the loss of crucial information. Moreover, standard microscopy techniques are limited to two-dimensional (2D) imaging, neglecting volumetric data crucial for detailed tissue analysis. X-ray imaging offers a non-destructive alternative, using contrast agents to enhance soft tissue visibility and allowing further investigations without compromising the sample. However, recently developed modified x-ray stains require adjustment for specific tissues, presenting a new challenge. Hematein can be chemically modified through the incorporation of high atomic number metals to enhance contrast in x-ray Imaging, whereas eosin staining can be augmented by increasing its concentration and acidifying the samples tissue. In this study, we aimed to demonstrate the feasibility of applying both modified hematoxylin and eosin stains to the same specimen sequentially, using a washing step with ethylenediaminetetraacetic acid (EDTA) to remove the hematein stain between scans. This novel approach preserves the distinct information provided by each stain, enabling comprehensive visualization in two separate micro-computed tomography (microCT) scans. The method was applied to biological samples from a rat strain spontaneously developing multiple endocrine tumors (MENX), provided by the Division of Neuroendocrinology at the Helmholtz Centre Munich. Specifically, the pituitary and adrenal glands of wild-type and MENX-affected rats were stained and imaged using the microCT system versaXRM-500 (ZEISS/xradia, Oberkochen Germany). The results revealed promising differentiation between healthy and affected tissues, with high-resolution imaging showing visible tumor formations, blood pools, and tissue degradation in diseased samples. This study highlights the potential of combining sequential H&E staining with microCT for enhanced tissue analysis and visualization of disease progression.
Grating-based x-ray phase-contrast imaging provides three simultaneous image channels originating from a single image acquisition. While the phase signal provides direct access to the electron density in tomography, there is additional information on sub-resolutional structural information which is called dark-field signal in analogy to optical microscopy. The additional availability of the conventional attenuation image qualifies the method for implementation into existing diagnostic routines. The simultaneous access to the attenuation coefficient and the electron density allows for quantitative two-material discrimination as demonstrated lately for measurements at a quasi-monochromatic compact synchrotron source. Here, we investigate the transfer of the method to conventional polychromatic x-ray sources and the additional inclusion of the dark-field signal for three-material decomposition. We evaluate the future potential of grating-based x-ray phase-contrast CT for quantitative three-material discrimination for the specific case of early stroke diagnosis at conventional polychromatic x-ray sources. Compared to conventional CT, the method has the potential to discriminate coagulated blood directly from contrast agent extravasation within a single CT acquisition. Additionally, the dark-field information allows for the clear identification of hydroxyapatite clusters due to their micro-structure despite a similar attenuation as the applied contrast agent. This information on materials with sub-resolutional microstructures is considered to comprise advantages relevant for various pathologies.
