Phase-contrast X-ray Tomographic Microscopy Research Articles

Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

3D Microstructure Reconstruction of Bamboo Fiber and Parenchyma Cell

ABSTRACT Synchrotron radiation absorption based and phase-contrast X-ray tomographic microscopy techniques are applied for observation and identification of the 3D features of bamboo fiber and parenchyma cells at micro-scale. The results revealed the 3D features within the fiber-reinforced bamboo. As to the 3D microstructure, the fiber and the parenchyma cells matrix are reconstructed, respectively. The 3D reconstruction will reveal necessary 3D geometrical and material parameters for better understanding the mechanical and biological functions of bamboo and provide new research ideas for relevant mechanism and the design of hierarchical structure of composite material.

Should We Ignore What We Cannot Measure? How Non-Uniform Stretch, Non-Uniform Wall Thickness and Minor Side Branches Affect Computational Aortic Biomechanics in Mice.

In order to advance the state-of-the-art in computational aortic biomechanics, we investigated the influence of (i) a non-uniform wall thickness, (ii) minor aortic side branches and (iii) a non-uniform axial stretch distribution on the location of predicted hotspots of principal strain in a mouse model for dissecting aneurysms. After 3 days of angiotensin II infusion, a murine abdominal aorta was scanned in vivo with contrast-enhanced micro-CT. The animal was subsequently sacrificed and its aorta was scanned ex vivo with phase-contrast X-ray tomographic microscopy (PCXTM). An automatic morphing framework was developed to map the non-pressurized, non-stretched PCXTM geometry onto the pressurized, stretched micro-CT geometry. The output of the morphing model was a structural FEM simulation where the output strain distribution represents an estimation of the wall deformation, not only due to the pressurization, but also due to the local axial stretch field. The morphing model also included minor branches and a mouse-specific wall thickness. A sensitivity study was then performed to assess the influence of each of these novel features on the outcome of the simulations. The results were supported by comparing the computed hotspots of principal strain to hotspots of early vascular damage as detected on PCXTM. Non-uniform axial stretch, non-uniform wall thickness and minor subcostal arteries significantly alter the locations of calculated hotspots of maximal principal strain. Even if experimental data on these features are often not available in clinical practice, one should be aware of the important implications that simplifications in the model might have on the final simulated result.

Angiotensin II infusion into ApoE-/- mice: a model for aortic dissection rather than abdominal aortic aneurysm?

Angiotensin II-infused ApoE-/- mice are a popular mouse model for preclinical aneurysm research. Here, we provide insight in the often-reported but seldom-explained variability in shape of dissecting aneurysms in these mice. N = 45 excised aortas were scanned ex vivo with phase-contrast X-ray tomographic microscopy. Micro-ruptures were detected near the ostium of celiac and mesenteric arteries in 8/11 mice that were sacrificed after 3 days of angiotensin II-infusion. At later time points (after 10, 18, and 28 days) the variability in shape of thoraco-abdominal lesions (occurring in 31/34 mice) was classified into 7 different categories based on the presence or absence of a medial tear (31/31), an intramural hematoma (23/31) or a false channel (11/23). Medial tears were detected both in the thoracic and the abdominal aorta and were most prevalent at the left and ventral aspects of celiac and mesenteric arteries. The axial length of the hematoma strongly correlated to the total number of ruptured branch ostia (r2 = 0.78) and in 22/23 mice with a hematoma the ostium of the left suprarenal artery had ruptured. Supraceliac diameters at baseline were significantly lower for mice that did not develop an intramural hematoma, and the formation of a false channel within that intramural hematoma depended on the location, rather than the length, of the medial tear. Based on our observations we propose an elaborate hypothesis that explains how aortic side branches (i) affect the initiation and propagation of medial tears and the subsequent adventitial dissection and (ii) affect the variability in shape of dissecting aneurysms. This hypothesis was partially validated through the live visualization of a dissecting aneurysm that formed during micro-CT imaging, and led us to the conclusion that angiotensin II-infused mice are more clinically relevant for the study of aortic dissections than for the study of abdominal aortic aneurysms.

Abstract 474: Early Events in Dissecting Aneurysms Induced by Angiotensin-II Infusion: The Geometry-Driven Aspect of a Multifaceted Problem

While research on dissecting aneurysms in Angiotensin-II infused mice spans more than a decade, the temporal sequence of initial events still remains unclear. Recent findings in our group suggested that focal medial tears at the vicinity of suprarenal side branches are the primary event in disease formation. In this study we used a combined experimental-computational approach to investigate the hypothesis that initial events of dissecting AAAs originate at branching sites along the aorta. Male apolipoprotein-deficient mice were infused with Angiotensin-II (n=11) and saline 0.9% (n=6) for 3 days and scanned with contrast-enhanced microCT prior to sacrifice. One animal presented an in-vivo rupture during the microCT scan, and was rescanned after 2.5 hours to observe real-time morphological changes. In all other animals, the excised aortic tissue was imaged with Phase Contrast X-Ray Tomographic Microscopy (PCXTM) at 6.5um isotropic resolution. An automatic morphing code was developed to map the ex-vivo geometry onto the in vivo geometry, and a finite element simulation yielded a stress distribution that represents an estimation of the wall tension, not only due to the pressurization, but also due to the local stretch field. We found that the nanoparticulate microCT contrast agent had infiltrated the aortic wall in 11/11 Ang-II infused animals, while no infiltration was observed in 6/6 control mice. The infiltration affected at least one pair of intercostal arteries in 11/11 mice, and in 9/11 mice the coeliac region was also affected. Image-guided histology allowed us to determine the circumferential distribution of microlesions at branching sites, including disruption of elastin fibers, apoptotic cell appearance, subintimal leukocyte infiltration and intramural hematomas. In the animal whose aorta had ruptured during the in vivo scan, the initial hematoma had originated around 3 pairs of intercostal arteries and quickly propagated afterwards. Mouse-specific finite element simulations revealed a co-location of computed peak stresses at the vessel wall and histologically identified vascular damage. We conclude that the aortic geometry, and side branches in particular, play a pivotal role in the onset of dissecting AAA.

Abstract 473: Dissecting Aortic Aneurym in Angiotensin II - Infused Mice: The Mechanisms Behind Lesion Variability

Angiotensin II-infused mice develop dissecting aneurysms, characterized by intramural rather than intraluminal thrombus, suprarenal rather than infrarenal lesions, medial dissections rather than circumferential elastin degradation and a variability in lesion shape that remained as yet unexplained. In order to provide insight into these intriguing lesions we scanned murine aortas at baseline and after 10, 18 and 29 days of Angiotensin II infusion, both in vivo (ultrasound, micro-CT) and ex vivo (phase-contrast X-ray tomographic microscopy). Dissecting aneurysms of varying severity occurred in 31/34 mice. All of these were characterized by a medial tear near the ostium of thoracic and abdominal aortic side branches, with a predilection for the left and ventral aspects of the ostium of the celiac artery. In 25/31 animals an intramural hematoma was formed. Fully ruptured branch ostia occurred significantly more often in the supraceliac aorta, affecting in particular the left suprarenal artery (the first branch cranial to the celiac artery, 23/25). Animals with a thoracic tear (6/31) had significantly larger intramural hematoma than animals with an abdominal tear (p<0.05), and the length of the hematoma correlated to the number of ruptured side branches (r 2 =0.78). In 11 mice a parallel false channel was formed. The volume of free-flowing intramural blood in the false channel was significantly larger for left than for ventral tears, but was not related to the length of the tear. Our data suggest that (i) medial tears are the primary event in dissecting AAA formation, (ii) an intramural hematoma is formed if the adventitia covering the medial tear dissects and leads to the accumulation of intramural blood from ruptured side branches, (iii) adventitial dissection and hematoma formation progress in the direction of least resistance/smallest side branches (i.e. cranial of the celiac artery) and (iv) a false channel is formed if the radial expansion of the adventitia due to blood flowing out of the medial tear acts in the same (leftward) direction as the expansion due to a ruptured left suprarenal artery. We conclude that Ang II-infused mice can be a valuable model to study the under-researched role of side branches in the formation and progression of aortic dissections.

Ascending Aortic Aneurysm in Angiotensin II–Infused Mice

Objective— To understand the anatomy and physiology of ascending aortic aneurysms in angiotensin II–infused ApoE −/− mice. Approach and Results— We combined an extensive in vivo imaging protocol (high-frequency ultrasound and contrast-enhanced microcomputed tomography at baseline and after 3, 10, 18, and 28 days of angiotensin II infusion) with synchrotron-based ultrahigh resolution ex vivo imaging (phase contrast X-ray tomographic microscopy) in n=47 angiotensin II–infused mice and 6 controls. Aortic regurgitation increased significantly over time, as did the luminal volume of the ascending aorta. In the samples that were scanned ex vivo, we observed one or several focal dissections, with the largest located in the outer convex aspect of the ascending aorta. The volume of the dissections moderately correlated to the volume of the aneurysm as measured in vivo ( r 2 =0.46). After 3 days of angiotensin II infusion, we found an interlaminar hematoma in 7/12 animals, which could be linked to an intimal tear. There was also a significant increase in single laminar ruptures, which may have facilitated a progressive enlargement of the focal dissections over time. At later time points, the hematoma was resorbed and the medial and adventitial thickness increased. Fatal transmural dissection occurred in 8/47 mice at an early stage of the disease, before adventita remodeling. Conclusions— We visualized and quantified the dissections that lead to ascending aortic aneurysms in angiotensin II–infused mice and provided unique insight into the temporal evolution of these lesions.

Dissecting abdominal aortic aneurysm in Ang II-infused mice: suprarenal branch ruptures and apparent luminal dilatation.

In this work, we provide novel insight into the morphology of dissecting abdominal aortic aneurysms in angiotensin II-infused mice. We demonstrate why they exhibit a large variation in shape and, unlike their human counterparts, are located suprarenally rather than infrarenally. We combined synchrotron-based, ultra-high resolution ex vivo imaging (phase contrast X-Ray tomographic microscopy) with in vivo imaging (high-frequency ultrasound and contrast-enhanced micro-CT) and image-guided histology. In all mice, we observed a tear in the tunica media of the abdominal aorta near the ostium of the celiac artery. Independently we found that, unlike the gradual luminal expansion typical for human aneurysms, the outer diameter increase of angiotensin II-induced dissecting aneurysms in mice was related to one or several intramural haematomas. These were caused by ruptures of the tunica media near the ostium of small suprarenal side branches, which had never been detected by the established small animal imaging techniques. The tear near the celiac artery led to apparent luminal dilatation, while the intramural haematoma led to a dissection of the tunica adventitia on the left suprarenal side of the aorta. The number of ruptured branches was higher in those aneurysms that extended into the thoracic aorta, which explained the observed variability in aneurysm shape. Our results are the first to describe apparent luminal dilatation, suprarenal branch ruptures, and intramural haematoma formation in dissecting abdominal aortic aneurysms in mice. Moreover, we validate and demonstrate the vast potential of phase contrast X-ray tomographic microscopy in cardiovascular small animal applications.

Micro-Scale Restraint Methodology for Humidity Induced Swelling Investigated by Phase Contrast X-Ray Tomography

A new methodology for restraining the swelling of spruce wood samples in the micrometre range is developed and presented. We show that the restraining device successfully prevents the free swelling of wood during moisture adsorption, thus modifying significantly the anisotropy of swelling and provoking the intended collapse and large deformations of the wood cells at the edges of the sample in contact with the restraining device. The device consists in a slotted cube designed to restrain swelling and is made of PMMA manufactured by laser ablation. The sample undergoing the restraining experiment is imaged with high-resolution synchrotron radiation phase contrast X-Ray Tomographic Microscopy. The deformation of the restraining device itself is only approximately 2 μm with respect to a 500 μm width in cubes containing latewood samples and half of that in the case of cubes containing earlywood.

Four-Dimensional Morphological Evolution of an Aluminum Silicon Alloy Using Propagation-Based Phase Contrast X-ray Tomographic Microscopy

Four-dimensional propagation-based phase contrast X-ray tomographic microscopy experiments were performed on an Aluminum29.9mass% Silicon alloy during coarsening. Using propagation-based phase contrast, changes in the three-dimensional morphology of primary silicon particles were captured and the resulting evolution of the microstructure is discussed. While morphologies at earlier times are complex, faceted and highly interconnected, the morphologies at later times are less faceted but remain quite complex.

Hysteresis in swelling and in sorption of wood tissue

The swelling and shrinkage of four Picea abies (L. Karst) wood tissue homogeneous samples, of porosity varying between 45% and 78%, is documented with high-resolution synchrotron radiation phase-contrast X-ray tomographic microscopy. We report measurements of the reversible moisture-induced orthotropic swelling/shrinkage strains. Hysteresis is observed when the swelling/shrinkage strain is considered as a function of relative humidity, except for the very high porosity sample. Hysteresis is no longer present when swelling/shrinkage strains are considered versus moisture content, indicating that wood deforms to the same extent whether an amount of moisture is desorbed or adsorbed. Furthermore, swelling anisotropy, in the tangential and radial directions, is found to increase with increasing porosity. The most homogeneous behaviour for a group of cells is found for 30-50 cells, smaller/larger groups having higher orders of variations.

Hysteretic swelling of wood at cellular scale probed by phase-contrast X-ray tomography

We investigated the three-dimensional, microscopic, dimensional changes of Picea abies (L. Karst) wood samples due to controlled steps of the ambient relative humidity. The study was performed at the wood cellular scale by high-resolution synchroton radiation phase-contrast X-ray tomographic microscopy (srPCXTM). Tomographic images were taken after the samples achieved moisture equilibrium at five adsorption and four desorption steps. For spruce latewood, swelling and shrinkage are found to be larger, more hysteretic and more homomorphic than for earlywood. Furthermore, while latewood undergoes similar strains in the transverse directions, earlywood radial strains are less than a third of the tangential strains. The less homomorphic and smaller swelling/shrinkage of earlywood in radial direction is found to be caused by the presence of rays.

3D imaging of microstructure of spruce wood

Synchrotron radiation phase-contrast X-ray tomographic microscopy (srPCXTM) was applied to observation and identification of the features of spruce anatomy at the cellular lengthscale. The pilot experiments presented in the paper clearly revealed the features of the heartwood of Spruce ( Picea abies [L.] Karst.), such as lumina and pits connecting the lumina, with a theoretical voxel size of 0.7 × 0.7 × 0.7 μm 3. The experiments were carried out on microspecimens of heartwood, measuring approximately 200 by 200 micrometers in cross-section. The technique for production and preparation of wood microsamples was developed within the framework of this investigation. The total porosity of the samples was derived and the values of the microstructural parameters, such as the diameters of tracheid, cell wall thicknesses and pit diameters were assessed non-invasively. Microstructural features as thin/small as approximately 1.5 μm were revealed and reconstructed in 3D. It is suggested that the position of sub-voxel-sized features (such as position of tori in the bordered pit pairs) can be determined indirectly using watershed segmentation. Moreover, the paper discusses the practical issues connected with a pipelined phase-contrast synchrotron-based microtomography experiment and the possible future potentials of this technique in the domain of wood science.