Movement analysis of primate molar teeth under load using synchrotron X-ray microtomography.

Mammalian teeth have to sustain repetitive and high chewing loads without failure. Key to this capability is the periodontal ligament (PDL), a connective tissue containing a collagenous fibre network which connects the tooth roots to the alveolar bone socket and which allows the teeth to move when loaded. It has been suggested that rodent molars under load experience a screw-like downward motion but it remains unclear whether this movement also occurs in primates. Here we use synchroton micro-computed tomography paired with an axial loading setup to investigate the form-function relationship between tooth movement and the morphology of the PDL space in a non-human primate, the mouse lemur (Microcebus murinus). The loading behavior of both mandibular and maxillary molars showed a three-dimensional movement with translational and rotational components, which pushes the tooth into the alveolar socket. Moreover, we found a non-uniform PDL thickness distribution and a gradual increase in volumetric proportion of the periodontal vasculature from cervical to apical. Our results suggest that the PDL morphology may optimize the three-dimensional tooth movement to avoid high stresses under loading.

Ex vivo evaluation of an atherosclerotic human coronary artery via histology and high-resolution hard X-ray tomography.

Atherosclerotic arteries exhibit characteristic constrictions and substantial deviations from cylindrical shape. Therefore, determining the artery's cross-section along the centerline is challenging, although high-resolution isotropic three-dimensional data are available. Herein, we apply high-resolution computed tomography in absorption and phase to a plaque-containing human artery post-mortem, through the course of the preparation stages for histology. We identify the impact of paraffin embedding and decalcification on the artery lumen. For automatic extraction of lumen's cross-section along centerline we present a dedicated pipeline. Comparing fixated tissue before and after paraffin embedding gives rise to shape changes with lumen reduction to 50-80%. The histological slicing induces further deformations with respect to tomography. Data acquired after decalcification show debris unintentionally distributed within the vessel preventing the reliable automatic lumen segmentation. Comparing tomography of laboratory- and synchrotron-radiation-based X rays by means of joint histogram analysis leads us to conclude that advanced desktop tomography is capable of quantifying the artery's lumen as an essential input for blood flow simulations. The results indicate that the most reliable lumen quantification is achieved by imaging the non-decalcified specimen fixed in formalin, using phase contrast modality and a dedicated processing pipeline. This study focusses on a methodology to quantitatively evaluate diseased artery segments post-mortem and provides unique structural parameters on the treatment-induced local shrinkage, which will be the basis of future studies on the flow in vessels affected by constrictions.

Full-field structured-illumination super-resolution X-ray transmission microscopy.

Modern transmission X-ray microscopy techniques provide very high resolution at low and medium X-ray energies, but suffer from a limited field-of-view. If sub-micrometre resolution is desired, their field-of-view is typically limited to less than one millimetre. Although the field-of-view increases through combining multiple images from adjacent regions of the specimen, so does the required data acquisition time. Here, we present a method for fast full-field super-resolution transmission microscopy by structured illumination of the specimen. This technique is well-suited even for hard X-ray energies above 30 keV, where efficient optics are hard to obtain. Accordingly, investigation of optically thick specimen becomes possible with our method combining a wide field-of-view spanning multiple millimetres, or even centimetres, with sub-micron resolution and hard X-ray energies.

Evaluation of contrasting techniques for X-ray imaging of velvet worms (Onychophora).

Non-invasive imaging techniques like X-ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft-bodied invertebrates, remains understudied. Herein, we used synchrotron radiation-based X-ray micro-computed tomography to compare the effects of commonly used contrasting approaches on onychophorans - soft-bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation-based and differential phase-contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal-to-noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box-and-whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X-ray micro-computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora.

X-ray phase microtomography with a single grating for high-throughput investigations of biological tissue.

The high-throughput 3D visualisation of biological specimens is essential for studying diseases and developmental disorders. It requires imaging methods that deliver high-contrast, high-resolution volumetric information at short sample preparation and acquisition times. Here we show that X-ray phase-contrast tomography using a single grating can provide a powerful alternative to commonly employed techniques, such as high-resolution episcopic microscopy (HREM). We present the phase tomography of a mouse embryo in paraffin obtained with an X-ray single-grating interferometer at I13-2 Beamline at Diamond Light Source and discuss the results in comparison with HREM measurements. The excellent contrast and quantitative density information achieved non-destructively and without staining using a simple, robust setup make X-ray single-grating interferometry an optimum candidate for high-throughput imaging of biological specimens as an alternative for existing methods like HREM.

Experimental Realisation of High-sensitivity Laboratory X-ray Grating-based Phase-contrast Computed Tomography.

The possibility to perform high-sensitivity X-ray phase-contrast imaging with laboratory grating-based phase-contrast computed tomography (gbPC-CT) setups is of great interest for a broad range of high-resolution biomedical applications. However, achieving high sensitivity with laboratory gbPC-CT setups still poses a challenge because several factors such as the reduced flux, the polychromaticity of the spectrum, and the limited coherence of the X-ray source reduce the performance of laboratory gbPC-CT in comparison to gbPC-CT at synchrotron facilities. In this work, we present our laboratory X-ray Talbot-Lau interferometry setup operating at 40 kVp and describe how we achieve the high sensitivity yet unrivalled by any other laboratory X-ray phase-contrast technique. We provide the angular sensitivity expressed via the minimum resolvable refraction angle both in theory and experiment, and compare our data with other differential phase-contrast setups. Furthermore, we show that the good stability of our high-sensitivity setup allows for tomographic scans, by which even the electron density can be retrieved quantitatively as has been demonstrated in several preclinical studies.

AHA classification of coronary and carotid atherosclerotic plaques by grating-based phase-contrast computed tomography.

OBJECTIVES: To evaluate the potential of grating-based phase-contrast computed-tomography (gb-PCCT) to classify human carotid and coronary atherosclerotic plaques according to modified American Heart Association (AHA) criteria. METHODS: Experiments were carried out at a laboratory-based set-up consisting of X-ray tube (40 kVp), grating-interferometer and detector. Eighteen human carotid and coronary artery specimens were examined. Histopathology served as the standard of reference. Vessel cross-sections were classified as AHA lesion type I/II, III, IV/V, VI, VII or VIII plaques by two independent reviewers blinded to histopathology. Conservative measurements of diagnostic accuracies for the detection and differentiation of plaque types were evaluated. RESULTS: A total of 127 corresponding gb-PCCT/histopathology sections were analyzed. Based on histopathology, lesion type I/II was present in 12 (9.5 %), III in 18 (14.2 %), IV/V in 38 (29.9 %), VI in 16 (12.6 %), VII in 34 (26.8 %) and VIII in 9 (7.0 %) cross-sections. Sensitivity, specificity and positive and negative predictive value were ≥0.88 for most analyzed plaque types with a good level of agreement (Cohen's kappa = 0.90). Overall, results were better in carotid (kappa = 0.97) than in coronary arteries (kappa = 0.85). Inter-observer agreement was high with kappa = 0.85, p < 0.0001. CONCLUSIONS: These results indicate that gb-PCCT can reliably classify atherosclerotic plaques according to modified AHA criteria with excellent agreement to histopathology. KEY POINTS: • Different atherosclerotic plaque types display distinct morphological features in phase-contrast CT. • Phase-contrast CT can detect and differentiate AHA plaque types. • Calcifications caused streak artefacts and reduced sensitivity in type VI lesions. • Overall agreement was higher in carotid than in coronary arteries.

X-ray phase-contrast computed tomography of human coronary arteries.

OBJECTIVE: The objective of this study was to assess the potential of grating-based phase-contrast computed tomography (gb-PCCT) for the detection and characterization of human coronary artery disease in an experimental ex vivo validation study. MATERIALS AND METHODS: The study was approved by the institutional review board, and informed consent was obtained from all patients. Specimens were examined using a conventional low-coherence x-ray tube (40 kV) and a Talbot-Lau grating interferometer. Histopathologic assessment was used as the standard of reference. Signal characteristics of calcified, fibrous (FIB), and lipid-rich (LIP) tissue were visually and quantitatively assessed by phase-contrast Hounsfield units (HU). Conventional absorption-based HU values were also measured. Conservative measurements of diagnostic accuracy for the detection and differentiation of plaque components as well as quantitative measurements of vessel dimensions were obtained, and receiver operating characteristic curve analysis for plaque differentiation was performed. RESULTS: A total of 15 coronary arteries from 5 subjects were available for analysis (386 sections). Calcified, FIB, and LIP displayed distinct gb-PCCT signal criteria. The diagnostic accuracy of gb-PCCT was high with sensitivity, specificity, and negative and positive predictive values of 0.89 or greater for all plaque components with good interrater agreement (к ≥ 0.88). In addition, quantitative measurements of vessel dimensions in gb-PCCT were strongly correlated with measurements obtained from histopathology (Pearson R ≥ 0.86). Finally, phase-contrast Hounsfield units were superior to conventional HU in differentiating FIB and LIP (receiver operating characteristic analysis, 0.86 vs. 0.77, respectively; P < 0.05). CONCLUSIONS: In an ex vivo setting, gb-PCCT provides improved differentiation and quantification of coronary atherosclerotic plaque and may thus serve as a tool for nondestructive histopathology.

X-ray phase contrast tomography by tracking near field speckle.

X-ray imaging techniques that capture variations in the x-ray phase can yield higher contrast images with lower x-ray dose than is possible with conventional absorption radiography. However, the extraction of phase information is often more difficult than the extraction of absorption information and requires a more sophisticated experimental arrangement. We here report a method for three-dimensional (3D) X-ray phase contrast computed tomography (CT) which gives quantitative volumetric information on the real part of the refractive index. The method is based on the recently developed X-ray speckle tracking technique in which the displacement of near field speckle is tracked using a digital image correlation algorithm. In addition to differential phase contrast projection images, the method allows the dark-field images to be simultaneously extracted. After reconstruction, compared to conventional absorption CT images, the 3D phase CT images show greatly enhanced contrast. This new imaging method has advantages compared to other X-ray imaging methods in simplicity of experimental arrangement, speed of measurement and relative insensitivity to beam movements. These features make the technique an attractive candidate for material imaging such as in-vivo imaging of biological systems containing soft tissue.

Visualizing typical features of breast fibroadenomas using phase-contrast CT: an ex-vivo study.

BACKGROUND: Fibroadenoma is the most common benign solid breast lesion type and a very common cause for histologic assessment. To justify a conservative therapy, a highly specific discrimination between fibroadenomas and other breast lesions is crucial. Phase-contrast imaging offers improved soft-tissue contrast and differentiability of fine structures combined with the potential of 3-dimensional imaging. In this study we assessed the potential of grating-based phase-contrast CT imaging for visualizing diagnostically relevant features of fibroadenomas. MATERIALS AND METHODS: Grating-based phase-contrast CT was performed on six ex-vivo formalin-fixed breast specimens containing a fibroadenoma and three samples containing benign changes that resemble fibroadenomas using Talbot Lau interferometry and a polychromatic X-ray source. Phase-contrast and simultaneously acquired absorption-based 3D-datasets were manually matched with corresponding histological slices. The visibility of diagnostically valuable features was assessed in comparison with histology as the gold-standard. RESULTS: In all cases, matching of grating-based phase-contrast CT images and histology was successfully completed. Grating-based phase-contrast CT showed greatly improved differentiation of fine structures and provided accurate depiction of strands of fibrous tissue within the fibroadenomas as well as of the diagnostically valuable dilated, branched ductuli of the fibroadenomas. A clear demarcation of tumor boundaries in all cases was provided by phase- but not absorption-contrast CT. CONCLUSIONS: Pending successful translation of the technology to a clinical setting and considerable reduction of the required dose, the data presented here suggest that grating-based phase-contrast CT may be used as a supplementary non-invasive diagnostic tool in breast diagnostics. Phase-contrast CT may thus contribute to the reduction of false positive findings and reduce the recall and core biopsy rate in population-based screening. Phase-contrast CT may further be used to assist during histopathological workup, offering a 3D view of the tumor and helping to identify diagnostically valuable tissue sections within large tumors.

FMT-PCCT: hybrid fluorescence molecular tomography-x-ray phase-contrast CT imaging of mouse models.

The implementation of hybrid fluorescence molecular tomography (FMT) and X-ray computed tomography (CT) has been shown to be a necessary development, not only for combining anatomical with functional and molecular contrast, but also for generating optical images of high accuracy. FMT affords highly sensitive 3-D imaging of fluorescence bio-distribution, but in stand-alone form it offers images of low resolution. It was shown that FMT accuracy significantly improves by considering anatomical priors from CT. Conversely, CT generally suffers from low soft tissue contrast. Therefore utilization of CT data as prior information in FMT inversion is challenging when different internal organs are not clearly differentiated. Instead, we combined herein FMT with emerging X-ray phase-contrast CT (PCCT). PCCT relies on phase shift differences in tissue to achieve soft tissue contrast superior to conventional CT. We demonstrate for the first time FMT-PCCT imaging of different animal models, where FMT and PCCT scans were performed in vivo and ex vivo, respectively. The results show that FMT-PCCT expands the potential of FMT in imaging lesions with otherwise low or no CT contrast, while retaining the cost benefits of CT and simplicity of hybrid device realizations. The results point to the most accurate FMT performance to date.

Phase-contrast CT: qualitative and quantitative evaluation of atherosclerotic carotid artery plaque.

PURPOSE: To evaluate the potential of phase-contrast computed tomography (CT) for atherosclerotic plaque imaging in human carotid arteries in an experimental ex vivo study. MATERIALS AND METHODS: The study was approved by the institutional review board, and informed consent was obtained from the patients' relatives. Seven postmortem human carotid artery specimens were imaged at a laboratory setup by using a conventional x-ray tube and grating interferometer. After histologic processing, phase-contrast imaging and histopathologic data were matched. Characteristics of the necrotic core (NC) covered by a fibrous cap (FC), intraplaque hemorrhage (IPH), and calcifications (CAs) were established, and sensitivity, specificity, and accuracy of phase-contrast CT for plaque detection and the potential for accurate quantification were assessed. The Cohen κ and Pearson correlation coefficient R were used to determine the agreement between phase-contrast imaging and histopathologic findings for plaque characterization and correlation of quantitative plaque measurements, respectively. A difference with a P value of less than .05 was considered significant. RESULTS: Characteristic criteria were found in all analyzed plaque components. Applying these criteria, phase-contrast CT had a good sensitivity for the detection of the FC and NC, IPH, and CAs (all, >80%) and excellent specificity and accuracy (all, >90%), with good interreader agreement (κ ≥ 0.72, P < .0001). There were excellent correlations for quantitative measurements of FC, NC, and CAs between phase-contrast imaging and histopathologic findings (R ≥ 0.92). Interreader reproducibility was excellent, with an intraclass correlation coefficient of 0.98 or higher for all measurements. CONCLUSION: The results of this study indicate that ex vivo phase-contrast CT can help identify and quantify atherosclerotic plaque components, with excellent correlation to histopathologic findings. Although not yet applicable in vivo, phase-contrast CT may become a valuable tool to monitor atherosclerotic disease process noninvasively.

Evaluation of phase-contrast CT of breast tissue at conventional X-ray sources - presentation of selected findings.

BACKGROUND: Grating-based phase contrast computed tomography (PC-CT) at synchrotron radiation sources has been shown to provide improved visualization of breast tumors. However, broad clinical application of phase-contrast imaging will likely depend on transferring the technology to standard polychromatic X-ray sources. On the basis of selected findings, we demonstrate the potential of grating-based PC-CT using a conventional X-ray source. MATERIALS AND METHODS: Grating-based PC-CT of two ex-vivo formalin fixed breast specimens containing lobular carcinoma was conducted using a Talbot Lau interferometer run at a polychromatic X-ray source of 40kVp. Phase-contrast and absorption-based 3D-datasets of both specimens were simultaneously recorded. Radiological images were manually matched with corresponding histological sections. The visualization of selected histological findings in phase contrast was compared to absorption contrast. RESULTS: Grating-based PC-CT was able to depict the 3-dimensional structure of dilated ducts and high phase contrast was found as a correlate to thickened fibrous ductal walls. Differences in contrast between fibrous and less fibrous breast tissue were observed in phase- but not in absorption-contrast images. Furthermore, regions of low phase contrast correlated with the extension of compact tumor components. CONCLUSIONS: On the basis of selected findings, we show that grating-based PC-CT at a polychromatic X-ray source provides complementary information to conventional absorption contrast; albeit at lower spatial resolution than synchrotron-based imaging.

A model for sound absorption by spheroidal particles.

This paper describes a mathematical model for the scattering of acoustic waves in dispersions of prolate or oblate non-spherical particles. Based on fundamental equations of change for mass, momentum, and energy, wave equations are derived and solved in spheroidal coordinates. The examination of the boundary-value problem of an aligned spheroidal particle in a continuous medium, excited by a plane wave, leads to a description of the viscoinertial, thermal, and diffractive phenomena. The model is analogous to the Epstein-Carhart-Allegra-Hawley theory for spherical particles, and suggests itself for studying non-sphericity in the acoustic analysis of industrial dispersions.