• Cochlea imaging
• Micro-CT
• Phase contrast
• Spiral ganglion neurons
• Synchrotron radiation
• X-ray tomography

• Animals
• Cochlea/diagnostic imaging
• Cochlea/anatomy & histology
• Imaging, Three-Dimensional/methods
• Tomography, X-Ray Computed/methods
• Primates
• Rats
• Rodentia/anatomy & histology

• Georg-August-Universität Göttingen (1018)

• 3D imaging
• Mouse
• mouse
• neuroscience
• structure tensor
• tractography
• vervet monkey
• white matter

• Animals
• White Matter/diagnostic imaging
• Mice
• Corpus Callosum/diagnostic imaging
• Corpus Callosum/anatomy & histology
• Diffusion Magnetic Resonance Imaging/methods
• Imaging, Three-Dimensional/methods
• Brain/diagnostic imaging
• Brain/anatomy & histology
• Demyelinating Diseases/diagnostic imaging
• Demyelinating Diseases/pathology

• R118-A11472 Lundbeck Foundation
• A5657 Captital Region of Denmark Research Foundation
• 101044180 European Research Council
• R347-2020-2454 Lundbeck Foundation
• 9039-00370B Independent Research Fund Denmark
• U54 MH091657 NIMH NIH HHS
• A41354 Scleroseforeningen

• compact μ-CT
• heavy-metal staining
• multi-scale imaging
• propagation-based phase contrast
• synchrotron radiation
• three-dimensional histology of the cochlea
• x-ray computed-tomography
• x-ray phase-contrast tomography

• P50 DC015857 NIDCD NIH HHS
• R01 DC000188 NIDCD NIH HHS
• U24 DC020849 NIDCD NIH HHS

The enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.

To examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.

Six patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton^® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.

In the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.

The analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed.

• Enteric nervous system
• Immunohistochemistry
• Neuropathy
• Synchrotron
• Virtual histology
• X-ray phase-contrast nanotomography

• Enteric Nervous System/pathology
• Eosine Yellowish-(YS)
• Hematoxylin
• Humans
• Ileum/diagnostic imaging
• Ileum/surgery
• Myenteric Plexus
• Paraffin
• X-Rays

For three-dimensional observation of unstained bio-specimens using X-ray microscopy with computed tomography (CT), one main problem has been low contrast in X-ray absorption. Here we introduce paraffin-mediated contrast enhancement to visualize biopsy samples of mouse kidney using a laboratory-based X-tray microscope. Unlike conventional heavy-atom staining, paraffin-mediated contrast enhancement uses solid paraffin as a negative contrast medium to replace water in the sample. The medium replacement from water to paraffin effectively lowers the absorption of low-energy X-rays by the medium, which eventually enhances the absorption contrast between the medium and tissue. In this work, paraffin-mediated contrast enhancement with 8 keV laboratory X-rays was used to visualize cylindrical renal biopsies with diameters of about 0.5 mm. As a result, reconstructed CT images from 19.4 h of data collection achieved cellular-level resolutions in all directions, which provided 3D structures of renal corpuscles from a normal mouse and from a disease model mouse. These two structures with and without disease allowed a volumetric analysis showing substantial volume differences in glomerular subregions. Notably, this nondestructive method presents CT opacities reflecting elemental composition and density of unstained tissues, thereby allowing more unbiased interpretation on their biological structures.

• Covid-19
• cardiac tissue
• epidemiology
• global health
• human
• x-ray tomography

• Artificial Intelligence
• COVID-19/complications
• COVID-19/pathology
• Heart/diagnostic imaging
• Heart/virology
• Humans
• Imaging, Three-Dimensional
• Myocarditis/diagnostic imaging
• Myocarditis/etiology
• Myocarditis/pathology
• Myocarditis/virology
• Myocardium/pathology
• SARS-CoV-2/isolation & purification
• Synchrotrons
• Tomography, X-Ray Computed

• Max Planck School Matter to Life Bundesministerium für Bildung und Forschung
• 05K19MG2 Bundesministerium für Bildung und Forschung
• EXC 2067/1-390729940 Deutsche Forschungsgemeinschaft
• XHale,771883 H2020 European Research Council
• KFO311 (project Z2) Deutsche Forschungsgemeinschaft
• XHale H2020 European Research Council
• 771883 H2020 European Research Council
• Hanseatic League of Science

• X-ray micro/nano CT
• low-density materials
• noise correlation
• phase retrieval
• propagation-based phase contrast

• 3D histology
• X-ray tomography
• biopsy
• pancreas
• phase retrieval

• Anisotropy
• Biopsy
• Humans
• Imaging, Three-Dimensional/instrumentation
• Microscopy, Phase-Contrast/instrumentation
• Pancreas/diagnostic imaging
• Pancreatic Neoplasms/diagnostic imaging
• Pancreatic Neoplasms/pathology
• Proof of Concept Study
• Tomography, X-Ray Computed/instrumentation
• User-Computer Interface

• 05K19MG2 Bundesministerium für Bildung und Forschung
• EXC2067 Deutsche Forschungsgemeinschaft

• X-ray imaging
• holography
• phase retrieval
• propagation-based phase contrast
• tomography

• SFB755 Deutsche Forschungsgemeinschaft
• 05K19MMG2 Bundesministerium für Bildung und Forschung

Purpose: We present a phase-contrast x-ray tomography study of wild type C57BL/6 mouse hearts as a nondestructive approach to the microanatomy on the scale of the entire excised organ. Based on the partial coherence at a home-built phase-contrast μ-CT setup installed at a liquid metal jet source, we exploit phase retrieval and hence achieve superior image quality for heart tissue, almost comparable to previous synchrotron data on the whole organ scale.

Approach: In our work, different embedding methods and heavy metal-based stains have been explored. From the tomographic reconstructions, quantitative structural parameters describing the three-dimensional (3-D) architecture have been derived by two different fiber tracking algorithms. The first algorithm is based on the local gradient of the reconstructed electron density. By performing a principal component analysis on the local structure-tensor of small subvolumes, the dominant direction inside the volume can be determined. In addition to this approach, which is already well established for heart tissue, we have implemented and tested an algorithm that is based on a local 3-D Fourier transform.

Results: We showed that the choice of sample preparation influences the 3-D structure of the tissue, not only in terms of contrast but also with respect to the structural preservation. A heart prepared with the evaporation-of-solvent method was used to compare both algorithms. The results of structural orientation were very similar for both approaches. In addition to the determination of the fiber orientation, the degree of filament alignment and local thickness of single muscle fiber bundles were obtained using the Fourier-based approach.

Conclusions: Phase-contrast x-ray tomography allows for investigating the structure of heart tissue with an isotropic resolution below 10  μm. The fact that this is possible with compact laboratory instrumentation opens up new opportunities for screening samples and optimizing sample preparation, also prior to synchrotron beamtimes. Further, results from the structural analysis can help in understanding cardiovascular diseases or can be used to improve computational models of the heart.

• anisotropy
• cardiomyocytes
• degree of filament alignment
• heart tissue
• laboratory propagation-based phase contrast
• orientation

• anisotropy
• cardiomyocytes
• degree of filament alignment
• heart tissue
• laboratory propagation-based phase contrast
• orientation

Purpose: Recently, progress has been achieved in implementing phase-contrast tomography of soft biological tissues at laboratory sources. This opens up opportunities for three-dimensional (3-D) histology based on x-ray computed tomography (μ- and nanoCT) in the direct vicinity of hospitals and biomedical research institutions. Combining advanced x-ray generation and detection techniques with phase reconstruction algorithms, 3-D histology can be obtained even of unstained tissue of the central nervous system, as shown, for example, for biopsies and autopsies of human cerebellum. Depending on the setup, i.e., source, detector, and geometric parameters, laboratory-based tomography can be implemented at very different sizes and length scales.

We investigate the extent to which 3-D histology of neuronal tissue can exploit the cone-beam geometry at high magnification M using a nanofocus transmission x-ray tube (nanotube) with a 300 nm minimal spot size (Excillum), combined with a single-photon counting camera. Tightly approaching the source spot with the biopsy punch, we achieve high M≈101−102, high flux density, and exploit the superior efficiency of this detector technology.

Approach: Different nanotube configurations such as spot size and flux, M, as well as exposure time, Fresnel number, and coherence are varied and selected in view of resolution, field of view, and/or phase-contrast requirements.

Results: The data show that the information content for the cytoarchitecture is enhanced by the phase effect. Comparison of results to those obtained at a microfocus rotating-anode x-ray tomography setup with a high-resolution detector, i.e., in low-M geometry, reveals similar to slightly superior data quality for the nanotube setup. In addition to its compactness, reduced power consumption by a factor of 103, and shorter scan duration, the particular advantage of the nanotube setup also lies in its suitability for pixel detector technology, enabling an increased range of opportunities for applications in laboratory phase-contrast x-ray tomography.

Conclusions: The phase retrieval scheme utilized mixes amplitude and phase contrast, with results being robust with respect to reconstruction parameters. Structural information content is comparable to slightly superior to previous results achieved with a microfocus rotating-anode setup but can be obtained in shorter scan time. Beyond advantages as compactness, lowered power consumption, and flexibility, the nanotube setup’s scalability in view of the progress in pixel detector technology is particularly beneficial. Further progress is thus likely to bring 3-D virtual histology to the performance in scan time and throughput required for clinical practice in neuropathology.

• cone-beam geometry
• human cerebellum
• nanofocus x-ray source
• neuronal tissue
• phase-contrast tomography

• 3D rendering
• Germination
• Image segmentation
• Pansy seeds
• Tomography
• X-ray microscope

• CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II and Ceitec Nano+ project, CZ.02.01/0.0./.0.0./16_013/0001728 under the program OP RDE

• 3d neuronal cytoarchitecture
• Embedding media for contrast enhancement
• Laboratory-based x-ray imaging
• Phase-contrast x-ray tomography
• Synchrotron-based x-ray imaging

• Animals
• Cerebellum/cytology
• Cerebellum/diagnostic imaging
• Imaging, Three-Dimensional/methods
• Mice
• Synchrotrons
• X-Ray Microtomography/methods

Spiders have evolved a unique male copulatory organ, the pedipalp bulb. The morphology of the bulb is species specific and plays an important role in species recognition and prezygotic reproductive isolation. Despite its importance for spider biodiversity, the mechanisms that control bulb development are virtually unknown. We have used confocal laser scanning microscopy (CLSM) and diffusible iodine-based contrast-enhanced micro computed tomography (dice-µCT) to study bulb development in the spider Parasteatoda tepidariorum. These imaging technologies enabled us to study bulb development in situ, without the use of destructive procedures for the first time. We show here that the inflated pedipalp tip in the subadult stage is filled with haemolymph that rapidly coagulates. Coagulation indicates histolytic processes that disintegrate tibia and tarsus, similar to histolytic processes during metamorphosis in holometabolous insects. The coagulated material contains cell inclusions that likely represent the cell source for the re-establishment of tarsus and tibia after histolysis, comparable to the histoblasts in insect metamorphosis. The shape of the coagulated mass prefigures the shape of the adult tarsus (cymbium) like a blueprint for the histoblasts. This suggests a unique role for controlled coagulation after histolysis in the metamorphosis-like morphogenesis of the male pedipalp.

• anatomy
• animal imaging
• computed tomography
• internal morphology
• μ‐CT

The complex cytoarchitecture of human brain tissue is traditionally studied by histology, providing structural information in 2D planes. This can be partly extended to 3D by inspecting many parallel slices, however, at nonisotropic resolution. This work shows that propagation-based X-ray phase-contrast tomography, both at the synchrotron and even at a compact laboratory source, can be used to perform noninvasive 3D virtual histology on unstained paraffin-embedded human cerebellum at isotropic subcellular resolution. The resulting data quality is high enough to visualize and automatically locate ∼10⁶ neurons within the different layers of the cerebellum, providing unprecedented data on its 3D cytoarchitecture and spatial organization.

To quantitatively evaluate brain tissue and its corresponding function, knowledge of the 3D cellular distribution is essential. The gold standard to obtain this information is histology, a destructive and labor-intensive technique where the specimen is sliced and examined under a light microscope, providing 3D information at nonisotropic resolution. To overcome the limitations of conventional histology, we use phase-contrast X-ray tomography with optimized optics, reconstruction, and image analysis, both at a dedicated synchrotron radiation endstation, which we have equipped with X-ray waveguide optics for coherence and wavefront filtering, and at a compact laboratory source. As a proof-of-concept demonstration we probe the 3D cytoarchitecture in millimeter-sized punches of unstained human cerebellum embedded in paraffin and show that isotropic subcellular resolution can be reached at both setups throughout the specimen. To enable a quantitative analysis of the reconstructed data, we demonstrate automatic cell segmentation and localization of over 1 million neurons within the cerebellar cortex. This allows for the analysis of the spatial organization and correlation of cells in all dimensions by borrowing concepts from condensed-matter physics, indicating a strong short-range order and local clustering of the cells in the granular layer. By quantification of 3D neuronal “packing,” we can hence shed light on how the human cerebellum accommodates 80% of the total neurons in the brain in only 10% of its volume. In addition, we show that the distribution of neighboring neurons in the granular layer is anisotropic with respect to the Purkinje cell dendrites.

• 3D virtual histology
• X-ray phase-contrast tomography
• automatic cell counting
• human brain cytoarchitecture

We demonstrate that phase retrieval and tomographic imaging at the organ level of small animals can be advantageously carried out using the monochromatic radiation emitted by a compact x-ray light source, without further optical elements apart from source and detector. This approach allows to carry out microtomography experiments which - due to the large performance gap with respect to conventional laboratory instruments - so far were usually limited to synchrotron sources. We demonstrate the potential by mapping the functional soft tissue within the guinea pig and marmoset cochlea, including in the latter case an electrical cochlear implant. We show how 3d microanatomical studies without dissection or microscopic imaging can enhance future research on cochlear implants.

Studies of brain cytoarchitecture in mammals are routinely performed by serial sectioning of the specimen and staining of the sections. The procedure is labor-intensive and the 3D architecture can only be determined after aligning individual 2D sections, leading to a reconstructed volume with non-isotropic resolution. Propagation-based x-ray phase-contrast tomography offers a unique potential for high-resolution 3D imaging of intact biological specimen due to the high penetration depth and potential resolution. We here show that even compact laboratory CT at an optimized liquid-metal jet microfocus source combined with suitable phase-retrieval algorithms and a novel tissue preparation can provide cellular and subcellular resolution in millimeter sized samples of mouse brain. We removed water and lipids from entire mouse brains and measured the remaining dry tissue matrix in air, lowering absorption but increasing phase contrast. We present single-cell resolution images of mouse brain cytoarchitecture and show that axons can be revealed in myelinated fiber bundles. In contrast to optical 3D techniques our approach does neither require staining of cells nor tissue clearing, procedures that are increasingly difficult to apply with increasing sample and brain sizes. The approach thus opens a novel route for high-resolution high-throughput studies of brain architecture in mammals.

• Micro-computed tomography
• X-ray imaging
• X-ray microscopy
• phase contrast
• phase retrieval

• Acoustic Stimulation
• Animals
• Channelrhodopsins
• Cochlea/physiopathology
• Cochlea/surgery
• Cochlear Implantation
• Deafness/surgery
• Electric Stimulation
• Evoked Potentials, Auditory
• Light
• Mice
• Mice, Inbred C57BL
• Mice, Transgenic
• Optogenetics
• Photic Stimulation
• Rats
• Rats, Transgenic
• Rats, Wistar
• Spiral Ganglion/pathology
• Spiral Ganglion/physiopathology

• X-ray imaging
• X-ray microscopy
• micro-tomography
• phase-contrast
• radiography

• x-ray phase contrast imaging
• x-ray tomography

• R01 CA142587 NCI NIH HHS
• R01 CA142587-02 NCI NIH HHS

• foraging
• fungal growth
• interactions
• invasions
• mycelia
• networks

• BB/E017312/1 Biotechnology and Biological Sciences Research Council