Many histological methods require staining of the cytoplasm, which provides instrumental details for diagnosis. One major limitation is the production of 2D images obtained by destructive preparation of 3D tissue samples. X-ray absorption micro- and nanocomputed tomography (microCT and nanoCT) allows for a nondestructive investigation of a 3D tissue sample, and thus aids to determine regions of interest for further histological examinations. However, application of microCT and nanoCT to biological samples (e.g., biopsies) is limited by the missing contrast within soft tissue, which is important to visualize morphological details. We describe an eosin-based preparation overcoming the challenges of contrast enhancement and selectivity for certain tissues. The eosin-based staining protocol is suitable for whole-organ staining, which then enables high-resolution microCT imaging of whole organs and nanoCT imaging of smaller tissue pieces retrieved from the original sample. Our results demonstrate suitability of the eosin-based staining method for diagnostic screening of 3D tissue samples without impeding further diagnostics through histological methods.

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of , a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Histological investigations are indispensable with regards to the identification of structural tissue details but are limited to two-dimensional images, which are often visualized in one and the same plane for comparison reasons. Nondestructive three-dimensional technologies such as X-ray micro- and nanoCT have proven to provide valuable benefits for the understanding of anatomical structures as they allow visualization of structural details in 3D and from arbitrary viewing angles. Nevertheless, low attenuation of soft tissue has hampered their application in the field of 3D virtual histology. We present a hematein-based X-ray staining method that specifically targets the cell nuclei of cells, as demonstrated for a whole liver lobule of a mouse. Combining the novel staining protocol with the high resolving power of a recently developed nanoCT system enables the 3D visualization of tissue architecture in the nanometer range, thereby revealing the real 3D morphology and spatial distribution of the cell nuclei. Furthermore, our technique is compatible with conventional histology, as microscopic slides can be derived from the very same stained soft-tissue sample and further counter staining is possible. Thus, our methodology demonstrates future applicability for modern histopathology using laboratory X-ray CT devices.

Dual-energy CT has opened up a new level of quantitative X-ray imaging for many diagnostic applications. The energy dependence of the X-ray attenuation is the key to quantitative material decomposition of the volume under investigation. This material decomposition allows the calculation of virtual native images in contrast enhanced angiography, virtual monoenergetic images for beam-hardening artifact reduction and quantitative material maps, among others. These visualizations have been proven beneficial for various diagnostic questions. Here, we demonstrate a new method of ‘virtual dual-energy CT’ employing grating-based phase-contrast for quantitative material decomposition. Analogue to the measurement at two different energies, the applied phase-contrast measurement approach yields dual information in form of a phase-shift and an attenuation image. Based on these two image channels, all known dual-energy applications can be demonstrated with our technique. While still in a preclinical state, the method features the important advantages of direct access to the electron density via the phase image, simultaneous availability of the conventional attenuation image at the full energy spectrum and therefore inherently registered image channels. The transfer of this signal extraction approach to phase-contrast data multiplies the diagnostic information gained within a single CT acquisition. The method is demonstrated with a phantom consisting of exemplary solid and fluid materials as well as a chicken heart with an iodine filled tube simulating a vessel. For this first demonstration all measurements have been conducted at a compact laser-undulator synchrotron X-ray source with a tunable X-ray energy and a narrow spectral bandwidth, to validate the quantitativeness of the processing approach.

Propagation-based imaging or inline holography in combination with computed tomography (holotomography) is a versatile tool to access a sample's three-dimensional (3D) micro or nano structure. However, the phase retrieval step needed prior to tomographic reconstruction can be challenging especially for strongly absorbing and refracting samples. Near-field ptychography is a recently developed phase imaging method that has been proven to overcome this hurdle in projection data. In this work we extend near-field ptychography to three dimensions and we show that, in combination with tomography, it can access the nano structure of a solid oxide fuel cell (SOFC). The quality of the resulting tomographic data and the structural properties of the anode extracted from this volume were compared to previous results obtained with holotomography. This work highlights the potential of 3D near-field ptychography for reliable and detailed investigations of samples at the nanometer scale, with important applications in materials and life sciences among others.