Phase contrast micro-CT with adjustable in-slice spatial resolution at constant magnification.

Objective.To report on a micro computed tomography (micro-CT) system capable of x-ray phase contrast imaging and of increasing spatial resolution at constant magnification.Approach.The micro-CT system implements the edge illumination (EI) method, which relies on two absorbing masks with periodically spaced transmitting apertures in the beam path; these split the beam into an array of beamlets and provide sensitivity to the beamlets' directionality, i.e. refraction. In EI, spatial resolution depends on the width of the beamlets rather than on the source/detector point spread function (PSF), meaning that resolution can be increased by decreasing the mask apertures, without changing the source/detector PSF or the magnification.Main results.We have designed a dedicated mask featuring multiple bands with differently sized apertures and used this to demonstrate that resolution is a tuneable parameter in our system, by showing that increasingly small apertures deliver increasingly detailed images. Phase contrast images of a bar pattern-based resolution phantom and a biological sample (a mouse embryo) were obtained at multiple resolutions.Significance.The new micro-CT system could find application in areas where phase contrast is already known to provide superior image quality, while the added tuneable resolution functionality could enable more sophisticated analyses in these applications, e.g. by scanning samples at multiple scales.

X-ray phase-contrast microtomography of soft tissues using a compact laboratory system with two-directional sensitivity.

X-ray microtomography is a nondestructive, three-dimensional inspection technique applied across a vast range of fields and disciplines, ranging from research to industrial, encompassing engineering, biology, and medical research. Phase-contrast imaging extends the domain of application of x-ray microtomography to classes of samples that exhibit weak attenuation, thus appearing with poor contrast in standard x-ray imaging. Notable examples are low-atomic-number materials, like carbon-fiber composites, soft matter, and biological soft tissues. We report on a compact and cost-effective system for x-ray phase-contrast microtomography. The system features high sensitivity to phase gradients and high resolution, requires a low-power sealed x-ray tube, a single optical element, and fits in a small footprint. It is compatible with standard x-ray detector technologies: in our experiments, we have observed that single-photon counting offered higher angular sensitivity, whereas flat panels provided a larger field of view. The system is benchmarked against known-material phantoms, and its potential for soft-tissue three-dimensional imaging is demonstrated on small-animal organs: a piglet esophagus and a rat heart. We believe that the simplicity of the setup we are proposing, combined with its robustness and sensitivity, will facilitate accessing quantitative x-ray phase-contrast microtomography as a research tool across disciplines, including tissue engineering, materials science, and nondestructive testing in general.

Technical note: Cartilage imaging with sub-cellular resolution using a laboratory-based phase-contrast x-ray microscope.

BACKGROUND: Microscopic imaging of cartilage is a key tool for the study and development of treatments for osteoarthritis. When cellular and sub-cellular resolution is required, histology remains the gold standard approach, albeit limited by the lack of volumetric information as well as by processing artifacts. Cartilage imaging with the sub-cellular resolution has only been demonstrated in the synchrotron environment. PURPOSE: To provide a proof-of-concept demonstration of the capability of a laboratory-based x-ray phase-contrast microscope to resolve sub-cellular features in a cartilage sample. METHODS: This work is based on a laboratory-based x-ray microscope using intensity-modulation masks. The structured nature of the beam, resulting from the mask apertures, allows the retrieval of three contrast channels, namely, transmission, refraction and dark-field, with resolution depending only on the mask aperture width. An ex vivo equine cartilage sample was imaged with the x-ray microscope and results were validated with synchrotron tomography and histology. RESULTS: Individual chondrocytes, that is, cells responsible for cartilage formation, could be detected with the laboratory-based microscope. The complementarity of the three retrieved contrast channels allowed the detection of sub-cellular features in the chondrocytes. CONCLUSIONS: We provide the first proof-of-concept of imaging cartilage tissue with sub-cellular resolution using a laboratory-based x-ray microscope.

Femtosecond multimodal imaging with a laser-driven X-ray source.

Laser-plasma accelerators are compact linear accelerators based on the interaction of high-power lasers with plasma to form accelerating structures up to 1000 times smaller than standard radiofrequency cavities, and they come with an embedded X-ray source, namely betatron source, with unique properties: small source size and femtosecond pulse duration. A still unexplored possibility to exploit the betatron source comes from combining it with imaging methods able to encode multiple information like transmission and phase into a single-shot acquisition approach. In this work, we combine edge illumination-beam tracking (EI-BT) with a betatron X-ray source and present the demonstration of multimodal imaging (transmission, refraction, and scattering) with a compact light source down to the femtosecond timescale. The advantage of EI-BT is that it allows multimodal X-ray imaging technique, granting access to transmission, refraction and scattering signals from standard low-coherence laboratory X-ray sources in a single shot.

Utility of knife-edge position tracking in cycloidal computed tomography.

Cycloidal computed tomography provides high-resolution images within relatively short scan times by combining beam modulation with dedicated under-sampling. However, implementing the technique relies on accurate knowledge of the sample's motion, particularly in the case of continuous scans, which is often unavailable due to hardware or software limitations. We have developed an easy-to-implement position tracking technique using a sharp edge, which can provide reliable information about the trajectory of the sample and thus improve the reconstruction process. Furthermore, this approach also enables the development of other innovative sampling schemes, which may otherwise be difficult to implement.

Volumetric High-Resolution X-Ray Phase-Contrast Virtual Histology of Breast Specimens With a Compact Laboratory System.

The assessment of margin involvement is a fundamental task in breast conserving surgery to prevent recurrences and reoperations. It is usually performed through histology, which makes the process time consuming and can prevent the complete volumetric analysis of large specimens. X-ray phase contrast tomography combines high resolution, sufficient penetration depth and high soft tissue contrast, and can therefore provide a potential solution to this problem. In this work, we used a high-resolution implementation of the edge illumination X-ray phase contrast tomography based on "pixel-skipping" X-ray masks and sample dithering, to provide high definition virtual slices of breast specimens. The scanner was originally designed for intra-operative applications in which short scanning times were prioritised over spatial resolution; however, thanks to the versatility of edge illumination, high-resolution capabilities can be obtained with the same system simply by swapping x-ray masks without this imposing a reduction in the available field of view. This makes possible an improved visibility of fine tissue strands, enabling a direct comparison of selected CT slices with histology, and providing a tool to identify suspect features in large specimens before slicing. Combined with our previous results on fast specimen scanning, this works paves the way for the design of a multi-resolution EI scanner providing intra-operative capabilities as well as serving as a digital pathology system.

Optimization of multipoint phase retrieval in edge illumination X-ray imaging: A theoretical and experimental analysis.

PURPOSE: In this work, an analytical model describing the noise in the retrieved three contrast channels, transmission, refraction, and ultra small-angle scattering, obtained with edge illumination X-ray phase-based imaging system is presented and compared to experimental data. METHODS: In EI, images acquired at different displacements of the presample mask (i.e., different illumination levels referred to as points on the "illumination curve"), followed by pixel-wise curve fitting, are exploited to quantitatively retrieve the three contrast channels. Therefore, the noise in the final image will depend on the error associated with the fitting process. We use a model based on the derivation of the standard error on fitted parameters, which relies on the calculation of the covariance matrix, to estimate the noise and the cross-channel correlation as a function of the position of the sampling points. In particular, we investigated the most common cases of 3 and 5 sampling points. In addition, simulations have been used to better understand the role of the integration time for each sampling point. Finally, the model is validated by comparison with the experimental data acquired with an edge illumination setup based on a tungsten rotating anode X-ray source and a photon counting detector. RESULTS: We found a good match between the predictions of the model and the experimental data. In particular, for the investigated cases, an arrangement of the sampling points leading to minimum noise and cross-channel correlation can be found. Simulations revealed that, given a fixed overall scanning time, its distribution into the smallest possible number of sampling points needed for phase retrieval leads to minimum noise thanks to higher statistics per point. CONCLUSIONS: This work presents an analytical model describing the noise in the various contrast channels retrieved in edge illumination as a function of the illumination curve sampling. In particular, an optimal sampling scheme leading to minimum noise has been determined for the case where 3 or 5 sampling points are used, which represent two of the most common acquisition schemes. In addition, the correlation between noise in the different channels and the role of the number of points and exposure time have been also investigated. In general, our results suggest a series of procedures that should be followed in order to optimize the experimental acquisitions.

Detection of involved margins in breast specimens with X-ray phase-contrast computed tomography.

Margins of wide local excisions in breast conserving surgery are tested through histology, which can delay results by days and lead to second operations. Detection of margin involvement intraoperatively would allow the removal of additional tissue during the same intervention. X-ray phase contrast imaging (XPCI) provides soft tissue sensitivity superior to conventional X-rays: we propose its use to detect margin involvement intraoperatively. We have developed a system that can perform phase-based computed tomography (CT) scans in minutes, used it to image 101 specimens approximately half of which contained neoplastic lesions, and compared results against those of a commercial system. Histological analysis was carried out on all specimens and used as the gold standard. XPCI-CT showed higher sensitivity (83%, 95% CI 69-92%) than conventional specimen imaging (32%, 95% CI 20-49%) for detection of lesions at margin, and comparable specificity (83%, 95% CI 70-92% vs 86%, 95% CI 73-93%). Within the limits of this study, in particular that specimens obtained from surplus tissue typically contain small lesions which makes detection more difficult for both methods, we believe it likely that the observed increase in sensitivity will lead to a comparable reduction in the number of re-operations.

Fast, non-iterative algorithm for quantitative integration of X-ray differential phase-contrast images.

X-ray phase contrast imaging is gaining importance as an imaging tool. However, it is common for X-ray phase detection techniques to be sensitive to the derivatives of the phase. Therefore, the integration of differential phase images is a fundamental step both to access quantitative pixel content and for further analysis such as segmentation. The integration of noisy data leads to artefacts with a severe impact on image quality and on its quantitative content. In this work, an integration method based on the Wiener filter is presented and tested using simulated and real data obtained with the edge illumination differential X-ray phase imaging method. The method is shown to provide high image quality while preserving the quantitative pixel content of the integrated image. In addition, it requires a short computational time making it suitable for large datasets.

A compact system for intraoperative specimen imaging based on edge illumination x-ray phase contrast.

A significant number of patients receiving breast-conserving surgery (BCS) for invasive carcinoma and ductal carcinoma in situ (DCIS) may need reoperation following tumor-positive margins from final histopathology tests. All current intraoperative margin assessment modalities have specific limitations. As a first step towards the development of a compact system for intraoperative specimen imaging based on edge illumination x-ray phase contrast, we prove that the system's dimensions can be reduced without affecting imaging performance. We analysed the variation in noise and contrast to noise ratio (CNR) with decreasing system length using the edge illumination x-ray phase contrast imaging setup. Two-(planar) and three-(computed tomography (CT)) dimensional imaging acquisitions of custom phantoms and a breast tissue specimen were made. Dedicated phase retrieval algorithms were used to separate refraction and absorption signals. A 'single-shot' retrieval method was also used, to retrieve thickness map images, due to its simple acquisition procedure and reduced acquisition times. Experimental results were compared to numerical simulations where appropriate. The relative contribution of dark noise signal in integrating detectors is significant for low photon count statistics acquisitions. Under constant exposure factors and magnification, a more compact system provides an increase in CNR. Superior CNR results were obtained for refraction and thickness map images when compared to absorption images. Results indicate that the 'single-shot' acquisition method is preferable for a compact CT intraoperative specimen scanner; it allows for shorter acquisition times and its combination of the absorption and refraction signals ultimately leads to a higher contrast. The first CT images of a breast specimen acquired with the compact system provided promising results when compared to those of the longer length system.

A Gaussian extension for Diffraction Enhanced Imaging.

Unlike conventional x-ray attenuation one of the advantages of phase contrast x-ray imaging is its capability of extracting useful physical properties of the sample. In particular the possibility to obtain information from small angle scattering about unresolvable structures with sub-pixel resolution sensitivity has drawn attention for both medical and material science applications. We report on a novel algorithm for the analyzer based x-ray phase contrast imaging modality, which allows the robust separation of absorption, refraction and scattering effects from three measured x-ray images. This analytical approach is based on a simple Gaussian description of the analyzer transmission function and this method is capable of retrieving refraction and small angle scattering angles in the full angular range typical of biological samples. After a validation of the algorithm with a simulation code, which demonstrated the potential of this highly sensitive method, we have applied this theoretical framework to experimental data on a phantom and biological tissues obtained with synchrotron radiation. Owing to its extended angular acceptance range the algorithm allows precise assessment of local scattering distributions at biocompatible radiation doses, which in turn might yield a quantitative characterization tool with sufficient structural sensitivity on a submicron length scale.

Large field of view, fast and low dose multimodal phase-contrast imaging at high x-ray energy.

X-ray phase contrast imaging (XPCI) is an innovative imaging technique which extends the contrast capabilities of 'conventional' absorption based x-ray systems. However, so far all XPCI implementations have suffered from one or more of the following limitations: low x-ray energies, small field of view (FOV) and long acquisition times. Those limitations relegated XPCI to a 'research-only' technique with an uncertain future in terms of large scale, high impact applications. We recently succeeded in designing, realizing and testing an XPCI system, which achieves significant steps toward simultaneously overcoming these limitations. Our system combines, for the first time, large FOV, high energy and fast scanning. Importantly, it is capable of providing high image quality at low x-ray doses, compatible with or even below those currently used in medical imaging. This extends the use of XPCI to areas which were unpractical or even inaccessible to previous XPCI solutions. We expect this will enable a long overdue translation into application fields such as security screening, industrial inspections and large FOV medical radiography - all with the inherent advantages of the XPCI multimodality.

Morphoproteomic Characterization of Lung Squamous Cell Carcinoma Fragmentation, a Histological Marker of Increased Tumor Invasiveness.

Accurate stratification of tumors is imperative for adequate cancer management. In addition to staging, morphologic subtyping allows stratification of patients into additional prognostic groups. In this study, we used an image-based computational method on pan-cytokeratin IHC stainings to quantify tumor fragmentation (TF), a measure of tumor invasiveness of lung squamous cell carcinoma (LSCC). In two independent clinical cohorts from tissue microarrays (TMA: n = 208 patients) and whole sections (WS: n = 99 patients), TF was associated with poor prognosis and increased risk of blood vessel infiltration. A third cohort from The Cancer Genome Atlas (TCGA: n = 335 patients) confirmed the poor prognostic value of TF using a similar human-based score on hematoxylin-eosin staining. Integration of RNA-seq data from TCGA and LC-MS/MS proteomics from WS revealed an upregulation of extracellular matrix remodeling and focal adhesion processes in tumors with high TF, supporting their increased invasive potential. This proposed histologic parameter is an independent and unfavorable prognostic marker that could be established as a new grading parameter for LSCC. Cancer Res; 77(10); 2585-93. ©2017 AACR.

Amyloid-ß plaque deposition measured using propagation-based X-ray phase contrast CT imaging.

Amyloid beta accumulation into insoluble plaques (Aßp) is known to play a significant role in the pathological process in Alzheimer's disease (AD). The presence of Aßp is also one of the neuropathological hallmarks for the disease. AD final diagnosis is generally acknowledged after the evaluation of Aßp deposition in the brain. Insoluble Aßp accumulation may also concur to cause AD as postulated in the so-called amyloid hypothesis. Therefore, the visualization, evaluation and quantification of Aßp are nowadays the keys for a better understanding of the disease, which may point to a possible cure for AD in the near future. Synchrotron-based X-ray phase contrast (XPC) has been demonstrated as the only imaging method that can retrieve the Aßp signal with high spatial resolution (up to 10 µm), high sensitivity and three-dimensional information at the same time. Although at the moment XPC is suitable for ex vivo samples only, it may develop into an alternative to positron emission tomography and magnetic resonance imaging in Aßp imaging. In this contribution the possibility of using synchrotron-based X-ray phase propagation computed tomography to visualize and measure Aßp on mouse brains is presented. A careful setup optimization for this application leads to a significant improvement of spatial resolution (∼1 µm), data acquisition speed (five times faster), X-ray dose (five times lower) and setup complexity, without a substantial loss in sensitivity when compared with the classic implementation of grating-based X-ray interferometry.

Asymmetric masks for laboratory-based X-ray phase-contrast imaging with edge illumination.

We report on an asymmetric mask concept that enables X-ray phase-contrast imaging without requiring any movement in the system during data acquisition. The method is compatible with laboratory equipment, namely a commercial detector and a rotating anode tube. The only motion required is that of the object under investigation which is scanned through the imaging system. Two proof-of-principle optical elements were designed, fabricated and experimentally tested. Quantitative measurements on samples of known shape and composition were compared to theory with good agreement. The method is capable of measuring the attenuation, refraction and (ultra-small-angle) X-ray scattering, does not have coherence requirements and naturally adapts to all those situations in which the X-ray image is obtained by scanning a sample through the imaging system.

A subcutaneous cellular implant for passive immunization against amyloid-ß reduces brain amyloid and tau pathologies.

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer's disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-ß peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-ß antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-ß antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer's pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-ß antibodies dramatically reduces amyloid-ß40 and amyloid-ß42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer's disease and other neurodegenerative disorders.

Ascending Aortic Aneurysm in Angiotensin II-Infused Mice: Formation, Progression, and the Role of Focal Dissections.

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.

Early post-metamorphic, Carboniferous blastoid reveals the evolution and development of the digestive system in echinoderms.

Inferring the development of the earliest echinoderms is critical to uncovering the evolutionary assembly of the phylum-level body plan but has long proven problematic because early ontogenetic stages are rarely preserved as fossils. Here, we use synchrotron tomography to describe a new early post-metamorphic blastoid echinoderm from the Carboniferous (approx. 323 Ma) of China. The resulting three-dimensional reconstruction reveals a U-shaped tubular structure in the fossil interior, which is interpreted as the digestive tract. Comparisons with the developing gut of modern crinoids demonstrate that crinoids are an imperfect analogue for many extinct groups. Furthermore, consideration of our findings in a phylogenetic context allows us to reconstruct the evolution and development of the digestive system in echinoderms more broadly; there was a transition from a straight to a simple curved gut early in the phylum's evolution, but additional loops and coils of the digestive tract (as seen in crinoids) were not acquired until much later.

Chronic Rho-kinase inhibition improves left ventricular contractile dysfunction in early type-1 diabetes by increasing myosin cross-bridge extension.

BACKGROUND: Impaired actin-myosin cross-bridge (CB) dynamics correlate with impaired left ventricular (LV) function in early diabetic cardiomyopathy (DCM). Elevated expression and activity of Rho kinase (ROCK) contributes to the development of DCM. ROCK targets several sarcomeric proteins including myosin light chain 2, myosin binding protein-C (MyBP-C), troponin I (TnI) and troponin T that all have important roles in regulating CB dynamics and contractility of the myocardium. Our aim was to examine if chronic ROCK inhibition prevents impaired CB dynamics and LV dysfunction in a rat model of early diabetes, and whether these changes are associated with changes in myofilament phosphorylation state. METHODS: Seven days post-diabetes induction (65 mg/kg ip, streptozotocin), diabetic rats received the ROCK inhibitor, fasudil (10 mg/kg/day ip) or vehicle for 14 days. Rats underwent cardiac catheterization to assess LV function simultaneous with X-ray diffraction using synchrotron radiation to assess in situ CB dynamics. RESULTS: Compared to controls, diabetic rats developed mild systolic and diastolic dysfunction, which was attenuated by fasudil. End-diastolic and systolic myosin proximity to actin filaments were significantly reduced in diabetic rats (P < 0.05). In all rats there was an inverse correlation between ROCK1 expression and the extension of myosin CB in diastole, with the lowest ROCK expression in control and fasudil-treated diabetic rats. In diabetic and fasudil-treated diabetic rats changes in relative phosphorylation of TnI and MyBP-C were not significant from controls. CONCLUSIONS: Our results demonstrate a clear role for ROCK in the development of LV dysfunction and impaired CB dynamics in early DCM.