Phase-contrast Micro-computed Tomography Research Articles

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

Multimodal Characterization of Nucleation and Progression of Interfacial Degradation in All Solid-State Batteries

We face an immediate need for more energy-dense batteries that are stable over long-term cycling, to address the increased electrification of the transportation sector. All-solid state batteries (ASSBs) that combine a solid-state electrolyte with a Li metal anode offer the potential to achieve this objective by replacing intercalation anodes such as graphite with Li metal. However, interfacial degradation at the Li | solid electrolyte interface currently compromises the safety and cycling stability of ASSBs [1]. This interfacial degradation is usually a combination of instabilities of mechanical, chemical or electrochemical origin. This in turn compromises the structural and morphological stability of ASSBs over cycling, causing them to fail in fewer cycles than required for implementation as next-generation batteries in automobiles [2]. Thus, in order to improve their cycling stability, the understanding of the origin and nature of these interfacial instabilities needs to be multimodal, to understand the interplay between the different degradation mechanisms. Argyrodite (Li6PS5Cl) is a particularly attractive solid-state electrolyte (SSE) due to a high ionic conductivity (comparable to liquid electrolytes), as well as potential for batch processing [3]. However, its brittle nature and chemical composition makes it susceptible to cracking and deleterious side reactions, which hamper its stability. This work will focus on elucidating the effect of (1) current density and (2) processing conditions on the cycling stability of Li | LPSCl | Li ASSBs.We use 4-D XRD-CT (X-ray diffraction computed tomography) combined with phase contrast micro-computed tomography (μCT) to conduct the multimodal investigation of interfacial degradation, under pseudo operando conditions. The methodology is to obtain a 3-D XRDCT scan around the interface in a particular cycled condition, followed by a higher-resolution 3D μCT scan on the same region. This sequence is repeated after every stripping/plating cycle, starting from the pristine cell up to cell failure. These experiments are conducted at a synchrotron source, to enable the acquisition at high spatial resolution (~5 μm) over large volumes (~mm3), in a reasonable amount of time. A standard swagelok-style cell is used for repeatability of results and optimal geometry for conducting tomography. XRD-CT can furnish quantitative phase maps of all phases (argyrodite and reaction by-products), as well as the elastic strain in the sample. The μCT on the other hand gives information on the evolution of morphology around the interface with cycling (cracks/voids). Thus, by correlating the two datasets together over cycling, we are able to connect mechanical instabilities to the chemical/electrochemical instabilities.Here, we focus on varying two specific processing parameters for argyrodite: the sintering temperature and pressure, and studying their effect on nucleation and propagation of interfacial instabilities over repeated cycling. Through cycling, we track the evolution of cracks/voids, chemical by-products such as LiCl, Li3P and Li2S using XRD-CT and μCT. Finally, we track the filling of metallic Li into certain cracks in dendritic form, which leads to shorting and failure of the cell. We find that both of these parameters heavily influence the behavior of the interface, with cells failing between 4-20 cycles, and a marked difference in how the degradation initiates and propagates. Finally, for the processing condition that shows the best cycling behavior, we do a C-rate study, by increasing the current density for plating/stripping with each cycle up to failure. This discusses the utility of a parameter such as critical current density for a cell, where the local current densities are very heterogeneous and different from the global current reading while cycling.We envisage these results being used as inputs into modeling studies to optimize strategies for interfacial stabilization in ASSBs going forward.[1] Paul, P. P. et al. Interfaces in all solid state Li-metal batteries: a review on instabilities, stabilization strategies, and scalability. Energy Storage Materials 2022, 45, 969-1001.[2] Hao, S. et al. Tracking lithium penetration in solid electrolytes in 3D by in-situ synchrotron X-ray computed tomography. Nano Energy 2021, 82, 105744.[3] Chen, Y-T. et al., Investigating dry room compatibility of sulfide solid-state electrolytes for scalable manufacturing, Journal of Materials Chemistry A 2022, 10, 7155-7164.

Abstract 13256: Efficient and Precise Phenotyping of Murine Fetal Hearts by Means of Synchrotron-Based Phase-Contrast Micro-Computed Tomography

Mouse models are routinely utilized to investigate the role of genes in congenital heart disease. Cardiovascular phenotyping is, however, quite challenging because of the small size of fetal mouse hearts. The most used method is histological assessment, which typically provides only two-dimensional information and thus might overlook important structural defects. Three-dimensional (3D) methods with sufficient resolution are either time-consuming, require specific sample preparation and/or selective staining, or cause destruction of the sample. Here, synchrotron radiation-based phase-contrast micro-computed tomography (SRμCT) was evaluated for fast and precise phenotyping of unstained, intact murine fetal hearts in a non-destructive way. Embryonic day (E) 18.5 hearts from control mice and mice that lack the secreted protease ADAMTS6 were formalin-fixed, paraffin-embedded and then scanned with SRμCT at the TOMCAT beamline (Swiss Light Source) with a 1.63 μm effective pixel size. Following SRμCT, the hearts were sectioned and processed for standard hematoxylin and eosin staining (H&E), and for specific immunofluorescence staining. SRμCT enabled evaluation of the hearts with a high level of detail. A full E18.5 mouse heart was scanned in less than 3 minutes, followed by virtual sectioning along arbitrary slicing planes and 3D rendering for morphological assessment. The resolution proved sufficient to trace minute features like the coronary arteries. In the Adamts6 knockouts, malformations such as a ventricular septal defect and double outlet right ventricle could be readily diagnosed. We also explored the potential of merging stained sections with SRμCT. In summary, SRμCT has the potential to become a powerful tool for fast phenotyping of mutant mouse models. The combination of 3D imaging with subsequent sectioning and methods like histology, immunohistochemistry and in situ hybridization will uncover pathophysiological mechanisms in 3D space.

Stem photosynthesis contributes to non-structural carbohydrate pool and modulates xylem vulnerability to embolism in Fraxinus ornus L.

Stem photosynthesis can significantly contribute to the carbon budget of woody plants, providing an extra carbon gain that might be crucial under drought stress causing leaf photosynthesis impairment and/or a reduced phloem transport.Stems of Fraxinus ornus L. saplings were covered with aluminum foil to test the impact of inhibition of stem photosynthesis on plant vulnerability to drought. Plants were water-stressed to target xylem water potential of −3.5 MPa and were then re-irrigated to field capacity to quantify their recovery capacity. Vulnerability to xylem embolism was assessed in light-exposed and stem-shaded saplings with both the hydraulic method and in vivo with X-ray phase contrast micro-computed tomography. We also measured non-structural carbohydrate (NSC) concentration and osmotic potential in bark and wood, separately.Stem shading increased xylem vulnerability to embolism formation under drought but did not influence the recovery phase. This difference was coupled with modification of the NSC pool and impaired osmoregulation, in particular in the wood of stem-shaded saplings compared to control ones.Our results indicate stem photosynthesis as an important source of local NSCs, directly or indirectly involved in osmoregulation processes, which could be crucial to enhance the hydraulic resistance to embolism formation and to endure drought.

Uterine leiomyoma as useful model to unveil morphometric and macromolecular collagen state and impairment in fibrotic diseases: An ex-vivo human study

Collagen is one of the main components of the extracellular matrix (ECM), involved, among all, in the maintenance of the structural support of tissues. In fibrotic diseases, collagen is overexpressed, and its production determines the formation of a significantly stiffer ECM. The cross-linking of high-resolution analytical tools, able to investigate both the tridimensional organization and the secondary structure of collagen in fibrotic diseases, could be useful to identify defined markers correlating the status of this protein with specific pathological conditions. To this purpose, an innovative multidisciplinary approach based on Phase-Contrast MicroComputed Tomography, Transmission Electron Microscopy, and Fourier Transform Infrared Imaging Spectroscopy was exploited on leiomyoma samples and adjacent myometrium to characterize microstructural collagen features. Uterine leiomyoma is a common gynecological disorder affecting women in fertile age. It is characterized by a massive collagen production due to the repairing processes occurring at myometrium level, and, hence, it represents a valuable model to investigate collagen self-organization in a pathological condition. Moreover, to evaluate the sensitivity of this multidisciplinary approach, the effects of eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids in collagen reduction were also investigated.

Interpreting Morphological Adaptations Associated with Viviparity in the Tsetse Fly Glossina morsitans (Westwood) by Three-Dimensional Analysis.

Simple SummaryTsetse flies, the sole transmitters of African Sleeping Sickness parasites, have a unique reproductive biology. They only develop one offspring at a time, they carry that offspring in their uterus for its entire immature development and provide nourishment for that offspring via milk-like secretions. This specialized reproductive biology has required dramatic modifications to the morphology of the reproductive organs in these and related flies. Here, we use phase contrast micro-Computed Tomography (Micro-CT) to visualize these adaptations in three dimensions for the first time. These adaptations include cuticular modifications allowing increased abdominal volume, expanded abdominal and uterine musculature, reduced egg development capacity, structural features of the male seminal secretions and detailed visualization of the gland responsible for synthesis and secretion of “milk” to feed intrauterine larvae. The ability to examine these tissues within the context of the rest of the organ systems in the fly provides new functional insights into how these changes have facilitated the evolution of the mating and reproductive biology of these flies.Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from most other insects, due to dramatic morphological and physiological adaptations required to support their unique biology. These adaptations are driven by demands associated with obligate hematophagy and viviparous reproduction. Obligate viviparity entails intrauterine larval development and the provision of maternal nutrients for the developing larvae. The reduced reproductive capacity/rate associated with this biology results in increased inter- and intra-sexual competition. Here, we use phase contrast microcomputed tomography (pcMicroCT) to analyze morphological adaptations associated with viviparous biology. These include (1) modifications facilitating abdominal distention required during blood feeding and pregnancy, (2) abdominal and uterine musculature adaptations for gestation and parturition of developed larvae, (3) reduced ovarian structure and capacity, (4) structural features of the male-derived spermatophore optimizing semen/sperm delivery and inhibition of insemination by competing males and (5) structural features of the milk gland facilitating nutrient incorporation and transfer into the uterus. Three-dimensional analysis of these features provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and provides new opportunities for comparative morphological analyses over time and between species.

Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level

High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. The highest quality images were obtained using propagation phase-contrast imaging and PTA-staining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p<0.0001 to p=0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

High-Resolution X-Ray Phase-Contrast 3-D Imaging of Breast Tissue Specimens as a Possible Adjunct to Histopathology.

Histopathological analysis is the current gold standard in breast cancer diagnosis and management, however, as imaging technology improves, the amount of potential diagnostic information that may be demonstrable radiologically should also increase. We aimed to evaluate the potential clinical usefulness of 3-D phase-contrast micro-computed tomography (micro-CT) imaging at high spatial resolutions as an adjunct to conventional histological microscopy. Ten breast tissue specimens, 2 mm in diameter, were scanned at the SYRMEP beamline of the Elettra Synchrotron using the propagation-based phase-contrast micro-tomography method. We obtained pixel size images, which were analyzed and compared with corresponding histological sections examined under light microscopy. To evaluate the effect of spatial resolution on breast cancer diagnosis, scans with four different pixel sizes were also performed. Our comparative analysis revealed that high-resolution images can enable, at a near-histological level, detailed architectural assessment of tissue that may permit increased breast cancer diagnostic sensitivity and specificity when compared with current imaging practices. The potential clinical applications of this method are also discussed.

Deformation of the Lamina Cribrosa and Optic Nerve Due to Changes in Cerebrospinal Fluid Pressure.

PurposeCerebrospinal fluid pressure (CSFp) changes are involved or implicated in various ocular conditions including glaucoma, idiopathic intracranial hypertension, and visual impairment and intracranial pressure syndrome. However, little is known about the effects of CSFp on lamina cribrosa and retrolaminar neural tissue (RLNT) biomechanics, potentially important in these conditions. Our goal was to use an experimental approach to visualize and quantify the deformation of these tissues as CSFp increased.MethodsThe posterior eye and RLNT of porcine eyes (n = 3) were imaged using synchrotron radiation phase-contrast micro-computed tomography (PC μCT) at an intraocular pressure of 15 mm Hg and CSFps of 4, 10, 20, and 30 mm Hg. Scans of each tissue region were acquired at each CSFp step and analyzed using digital volume correlation to determine 3-dimensional tissue deformations.ResultsElevating CSFp increased the strain in the lamina cribrosa and RLNT of all three specimens, with the largest strains occurring in the RLNT. Relative to the baseline CSFp of 4 mm Hg, at 30 mm Hg, the lamina cribrosa experienced a mean first and third principal strain of 4.4% and −3.5%, respectively. The corresponding values for the RLNT were 9.5% and −9.1%.ConclusionsCSFp has a significant impact on the strain distributions within the lamina cribrosa and, more prominently, within the RLNT. Elevations in CSFp were positively correlated with increasing deformations in each region and may play a role in ocular pathologies linked to changes in CSFp.

Phase-Contrast Micro-Computed Tomography Measurements of the Intraocular Pressure-Induced Deformation of the Porcine Lamina Cribrosa

The lamina cribrosa (LC) is a complex mesh-like tissue in the posterior eye. Its biomechanical environment is thought to play a major role in glaucoma, the second most common cause of blindness. Due to its small size and relative inaccessibility, high-resolution measurements of LC deformation, important in characterizing LC biomechanics, are challenging. Here we present a novel noninvasive imaging method, which enables measurement of the three-dimensional deformation of the LC caused by acute elevation of intraocular pressure (IOP). Posterior segments of porcine eyes were imaged using synchrotron radiation phase contrast micro-computed tomography (PC μCT) at IOPs between 6 and 37 mmHg. The complex trabecular architecture of the LC was reconstructed with an isotropic spatial resolution of 3.2 μm. Scans acquired at different IOPs were analyzed with digital volume correlation (DVC) to compute full-field deformation within the LC. IOP elevation caused substantial tensile, shearing and compressive devformation within the LC, with maximum tensile strains at 30 mmHg averaging 5.5%, and compressive strains reaching 20%. We conclude that PC μCT provides a novel high-resolution method for imaging the LC, and when combined with DVC, allows for full-field 3D measurement of ex vivo LC biomechanics at high spatial resolution.

Synchrotron- and laboratory-based X-ray phase-contrast imaging for imaging mouse articular cartilage in the absence of radiopaque contrast agents

The mouse model of osteoarthritis (OA) has been recognized as the most promising research tool for the identification of new OA therapeutic targets. However, this model is currently limited by poor throughput, dependent on the extremely time-consuming histopathology assessment of the articular cartilage (AC). We have recently shown that AC in the rat tibia can be imaged both in air and in saline solution using a laboratory system based on coded-aperture X-ray phase-contrast imaging (CAXPCi). Here, we explore ways to extend the methodology for imaging the much thinner AC of the mouse, by means of gold-standard synchrotron-based phase-contrast methods. Specifically, we have used analyser-based phase-contrast micro-computed tomography (micro-CT) for its high sensitivity to faint phase changes, coupled with a high-resolution (4.5 μm pixel) detector. Healthy, diseased (four weeks post induction of OA) and artificially damaged mouse AC was imaged at the Elettra synchrotron in Trieste, Italy, using the above method. For validation, we used conventional micro-CT combined with radiopaque soft-tissue staining and standard histomorphometry. We show that mouse cartilage can be visualized correctly by means of the synchrotron method. This suggests that: (i) further developments of the laboratory-based CAXPCi system, especially in terms of pushing the resolution limits, might have the potential to resolve mouse AC ex vivo and (ii) additional improvements may lead to a new generation of CAXPCi micro-CT scanners which could be used for in vivo longitudinal pre-clinical imaging of soft tissue at resolutions impossible to achieve by current MRI technology.

Three-dimensional imaging of whole mouse models: comparing nondestructive X-ray phase-contrast micro-CT with cryotome-based planar epi-illumination imaging

In this study, we compare two evolving techniques for obtaining high-resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome-based planar epi-illumination imaging (cryo-imaging). On the other hand, we examine X-ray phase-contrast micro-computed tomography (micro-CT) using synchrotron radiation. Cryo-imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo-frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X-ray phase-contrast imaging is based on the minute refraction of X-rays inside the specimen and features higher soft-tissue contrast than conventional, attenuation-based micro-CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft-tissue contrast is strongly enhanced in the X-ray phase images compared to the attenuation-based images. This identifies phase-contrast micro-CT as a powerful tool for the study of small animal disease models.

Pain, motor and gait assessment of murine osteoarthritis in a cruciate ligament transection model

The major complaint of Osteoarthritis (OA) patients is pain. However, due to the nature of clinical studies and the limitation of animal studies, few studies have linked function impairment and behavioral changes in OA animal models to cartilage loss and histopathology. Our objective was to study surrogate markers of functional impairment in relation to cartilage loss and pathological changes in a post-traumatic mouse model of OA. We performed a battery of functional analyses in a mouse model of OA generated by cruciate ligament transection (CLT). The changes in functional analyses were linked to histological changes graded by OARSI standards, histological grading of synovitis, and volumetric changes of the articular cartilage and osteophytes quantified by phase contrast micro-computed tomography (μCT). OA generated by CLT led to decreased time on rotarod, delayed response on hotplate analysis, and altered gait starting from 4 weeks after surgery. Activity in open field analysis did not change at 4, 8, or 12 weeks after CLT. The magnitude of behavioral changes was directly correlated with higher OARSI histological scores of OA, synovitis in the knee joints, cartilage volume loss, and osteophyte formation. Our findings link functional analyses to histological grading, synovitis, comprehensive three-dimensional assessment of cartilage volume and osteophyte formation. This serves as a reference for a mouse model in predicting outcomes of OA treatment.

Quantitative imaging of murine osteoarthritic cartilage by phase contrast micro-computed tomography

Objective—Mouse is an optimal model organism in which gene-environment interactions can be studied for the pathogenesis of osteoarthritis. The gold standard in arthritis research in mice is based on histopathology and immunohistochemistry, which are labor intensive, prone to sampling bias and technical variability, and limited in throughput. This study aims to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques. Methods—A novel mouse model of osteoarthritis was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint samples were then imaged by a novel technique that combines high resolution μ CT and phase-contrast optics followed by quantitative analyses. A comparative analysis was also performed on a previously established mouse model of osteoarthritis generated by destabilization of medial meniscus (DMM). Results—Phase contrast μ CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase contrast μ CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue levels could also be observed in images generated by phase contrast μ CT. Conclusion—We established an imaging technology that comprehensively assessed and quantified the 2 and 3 dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high throughput assessment of genetic and therapeutic models of OA in mice.

Phase Contrast Micro-Computed Tomography of Biological Sample at SSRF

In line X-ray phase contrast micro-computed tomography (IL-XPCT), which can be implemented at third generation synchrotron radiation sources or by using a micro-focus X-ray tube, is a powerful technique for non-destructive, high-resolution investigations of a broad variety of materials. At the Shanghai Synchrotron Radiation Facility (SSRF), the X-ray Imaging and Biomedical Applications Beamline was built and started regular user operation in May 2009. Both qualitative (without phase retrieval) and quantitative (with phase retrieval) three-dimensional IL-XPCT experimental techniques have been established at the beamline. IL-XPCT experiments of a test sample (plastic pipes) used to evaluate the technique, and of a biological sample (locust) at the beamline are reported. Two series of images, qualitative and quantitative, including tomographic slices and three-dimensional rendering images were obtained. In qualitative images, there is a strong edge-enhancement which leads to very clear sample contours, while in quantitative images, the edge-enhancement fades but quantitative measurement of sample's phase information could be achieved. The experiments demonstrate that the combination of qualitative and quantitative images is useful for biological sample studies.

Evaluation of ultrafast laser-based hard x-ray sources for phase-contrast imaging

The 2D projection phase-contrast imaging performance of the ultrafast laser-based x-ray (ULX) source has been investigated. The potential of such a novel x-ray source has been assessed by imaging a reference object (Contrast Detail Evaluation phantom) in the in-line holography geometry and by applying a simple 1D numerical model to the data analysis. The results indicate that the ULX is a promising technique for 2D projection phase-contrast imaging and for implementation of phase-contrast micro-Computed Tomography (μ-CT). This is because by using high contrast laser pulse ULX simultaneously provides a very small x-ray source size along with a high average x-ray flux. In addition, due to the ultrashort x-ray burst duration, ULX might allow practical implementation of ultrafast phase-contrast stroboscopy and time-of-flight based electronic scatter rejection. This technique is also of interest for time resolved radiography to follow shock waves and radiative fronts propagating in an opaque matter.