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.

Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Edge-illumination spectral phase-contrast tomography.

Following the rapid, but independent, diffusion of x-ray spectral and phase-contrast systems, this work demonstrates the first combination of spectral and phase-contrast computed tomography (CT) obtained by using the edge-illumination technique and a CdTe small-pixel (62µm) spectral detector. A theoretical model is introduced, starting from a standard attenuation-based spectral decomposition and leading to spectral phase-contrast material decomposition. Each step of the model is followed by quantification of accuracy and sensitivity on experimental data of a test phantom containing different solutions with known concentrations. An example of a micro CT application (20µm voxel size) on an iodine-perfusedex vivomurine model is reported. The work demonstrates that spectral-phase contrast combines the advantages of spectral imaging, i.e. high-Zmaterial discrimination capability, and phase-contrast imaging, i.e. soft tissue sensitivity, yielding simultaneously mass density maps of water, calcium, and iodine with an accuracy of 1.1%, 3.5%, and 1.9% (root mean square errors), respectively. Results also show a 9-fold increase in the signal-to-noise ratio of the water channel when compared to standard spectral decomposition. The application to the murine model revealed the potential of the technique in the simultaneous 3D visualization of soft tissue, bone, and vasculature. While being implemented by using a broad spectrum (pink beam) at a synchrotron radiation facility (Elettra, Trieste, Italy), the proposed experimental setup can be readily translated to compact laboratory systems including conventional x-ray tubes.

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.

Modulation transfer function (MTF) evaluation for x-ray phase imaging system employing attenuation masks.

Objective.Attenuation masks can be used in x-ray imaging systems to increase their inherent spatial resolution and/or make them sensitive to phase effects, a typical example being Edge Illumination x-ray phase contrast imaging (EI-XPCI). This work investigates the performance of a mask-based system such as EI-XPCI in terms of Modulation Transfer Function (MTF), in the absence of phase effects.Approach. Pre-sampled MTF measurements, using an edge, were performed on the same system implemented without masks, with non-skipped masks and finally with skipped masks (i.e. masks in which apertures illuminate every other pixel row/column). Results are compared to simulations and finally images of a resolution bar pattern acquired with all the above setups are presented.Main results. Compared to the detector's inherent MTF, the non-skipped mask setup provides improved MTF results. In comparison to an ideal case where signal spill-out into neighbouring pixels is negligible, this improvement takes place only at specific frequencies of the MTF, dictated by the spatial repetition of the spill-out signal. This is limited with skipped masks, which indeed provide further MTF improvements over a larger frequency range. Experimental MTF measurements are supported through simulation and resolution bar pattern images.Significance. This work has quantified the improvement in MTF due to the use of attenuation masks and lays the foundation for how acceptance and routine quality control tests will have to be modified when systems using masks are introduced in clinical practice and how MTF results will compare to those of conventional imaging systems.

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.

Freestanding high-aspect-ratio gold masks for low-energy, phase-based x-ray microscopy.

High-resolution, x-ray phase contrast microscopy, a key technique with promising potential in biomedical imaging and diagnostics, is based on narrow-slit high-aspect-ratio gold gratings. We present the development, fabrication details, and experimental testing of the freestanding 10µm thick gold membrane masks with an array of 0.9-1.5µm void slit apertures for a novel low-energy x-ray microscope. The overall mask size is 4 mm × 4 mm, with a grating pitch of 7.5µm, 6.0-6.6µm wide gold bars are supported by 3µm wide crosslinks at 400µm intervals. The fabrication process is based on gold electroplating into a silicon mold coated with various thin films to form a voltage barrier, plating base, and sacrificial layer, followed by the mold removal to obtain the freestanding gold membrane with void slit apertures. We discuss key aspects for the materials and processes, including gold structures homogeneity, residual stresses, and prevention of collapsing of the grid elements. We further demonstrate the possibility to obtain high-resolution, high contrast 2D images of biological samples using an incoherent, rotating anode x-ray tube.

Time resolved in-situ multi-contrast X-ray imaging of melting in metals.

In this work, the application of a time resolved multi-contrast beam tracking technique to the investigation of the melting and solidification process in metals is presented. The use of such a technique allows retrieval of three contrast channels, transmission, refraction and dark-field, with millisecond time resolution. We investigated different melting conditions to characterize, at a proof-of-concept level, the features visible in each of the contrast channels. We found that the phase contrast channel provides a superior visibility of the density variations, allowing the liquid metal pool to be clearly distinguished. Refraction and dark-field were found to highlight surface roughness formed during solidification. This work demonstrates that the availability of the additional contrast channels provided by multi-contrast X-ray imaging delivers additional information, also when imaging high atomic number specimens with a significant absorption.

A laboratory-based, low-energy, multi-modal x-ray microscope with user-defined resolution.

We report on the development of a low-energy x-ray phase-based microscope using intensity-modulation masks for single-shot retrieval of three contrast channels: transmission, refraction, and ultra-small-angle scattering or dark field. The retrieval method is based on beam tracking, an incoherent and phase-based imaging approach. We demonstrate that the spatial resolution of this imaging system does not depend on focal spot size nor detector pixel pitch, as opposed to conventional and propagation-based x-ray imaging, and it is only dependent on the mask aperture size. This result enables the development of a multi-resolution microscope where multi-scale samples can be explored on different length scales by adjusting only the mask aperture size, without other modifications. Additionally, we show an extended capability of the system to resolve periodic structures below the resolution limit imposed by the mask apertures, which potentially extends dark-field imaging beyond its conventional use.

Endoscopic eradication therapy for Barrett's esophagus-related neoplasia: a final 10-year report from the UK National HALO Radiofrequency Ablation Registry.

BACKGROUND AND AIMS: Long-term durability data for effectiveness of radiofrequency ablation (RFA) to prevent esophageal adenocarcinoma in patients with dysplastic Barrett's esophagus (BE) are lacking. METHODS: We prospectively collected data from 2535 patients with BE (mean length, 5.2 cm; range, 1-20) and neoplasia (20% low-grade dysplasia, 54% high-grade dysplasia, 26% intramucosal carcinoma) who underwent RFA therapy across 28 UK hospitals. We assessed rates of invasive cancer and performed detailed analyses of 1175 patients to assess clearance rates of dysplasia (CR-D) and intestinal metaplasia (CR-IM) within 2 years of starting RFA therapy. We assessed relapses and rates of return to CR-D (CR-D2) and CR-IM (CR-IM2) after further therapy. CR-D and CR-IM were confirmed by an absence of dysplasia and intestinal metaplasia on biopsy samples taken at 2 consecutive endoscopies. RESULTS: Ten years after starting treatment, the Kaplan-Meier (KM) cancer rate was 4.1% with a crude incidence rate of .52 per 100 patient-years. CR-D and CR-IM after 2 years of therapy were 88% and 62.6%, respectively. KM relapse rates were 5.9% from CR-D and 18.7% from CR-IM at 8 years, with most occurring in the first 2 years. Both were successfully retreated with rates of CR-D2 of 63.4% and CR-IM2 of 70.0% 2 years after retreatment. EMR before RFA increased the likelihood of rescue EMR from 17.2% to 41.7% but did not affect the rate of CR-D, whereas rescue EMR after RFA commenced reduced CR-D from 91.4% to 79.7% (χ2P < .001). CONCLUSIONS: RFA treatment is effective and durable to prevent esophageal adenocarcinoma. Most treatment relapses occur early and can be successfully retreated.

Cycloidal CT with CNN-based sinogram completion and in-scan generation of training data.

In x-ray computed tomography (CT), the achievable image resolution is typically limited by several pre-fixed characteristics of the x-ray source and detector. Structuring the x-ray beam using a mask with alternating opaque and transmitting septa can overcome this limit. However, the use of a mask imposes an undersampling problem: to obtain complete datasets, significant lateral sample stepping is needed in addition to the sample rotation, resulting in high x-ray doses and long acquisition times. Cycloidal CT, an alternative scanning scheme by which the sample is rotated and translated simultaneously, can provide high aperture-driven resolution without sample stepping, resulting in a lower radiation dose and faster scans. However, cycloidal sinograms are incomplete and must be restored before tomographic images can be computed. In this work, we demonstrate that high-quality images can be reconstructed by applying the recently proposed Mixed Scale Dense (MS-D) convolutional neural network (CNN) to this task. We also propose a novel training approach by which training data are acquired as part of each scan, thus removing the need for large sets of pre-existing reference data, the acquisition of which is often not practicable or possible. We present results for both simulated datasets and real-world data, showing that the combination of cycloidal CT and machine learning-based data recovery can lead to accurate high-resolution images at a limited dose.

Monitoring tissue engineered constructs and protocols with laboratory-based x-ray phase contrast tomography.

Tissue engineering (TE) aims to generate bioengineered constructs which can offer a surgical treatment for many conditions involving tissue or organ loss. Construct generation must be guided by suitable assessment tools. However, most current tools (e.g. histology) are destructive, which restricts evaluation to a single-2D anatomical plane, and has no potential for assessing constructs prior to or following their implantation. An alternative can be provided by laboratory-based x-ray phase contrast computed tomography (PC-CT), which enables the extraction of 3D density maps of an organ's anatomy. In this work, we developed a semi-automated image processing pipeline dedicated to the analysis of PC-CT slices of oesophageal constructs. Visual and quantitative (density and morphological) information is extracted on a volumetric basis, enabling a comprehensive evaluation of the regenerated constructs. We believe the presented tools can enable the successful regeneration of patient-specific oesophagus, and bring comparable benefit to a wide range of TE applications. STATEMENT OF SIGNIFICANCE: Phase contrast computed tomography (PC-CT) is an imaging modality which generates high resolution volumetric density maps of biological tissue. In this work, we demonstrate the use of PC-CT as a new tool for guiding the progression of an oesophageal tissue engineering (TE) protocol. Specifically, we developed a semi-automated image-processing pipeline which analyses the oesophageal PC-CT slices, extracting visual and quantitative (density and morphological) information. This information was proven key for performing a comprehensive evaluation of the regenerated constructs, and cannot be obtained through existing assessment tools primarily due to their destructive nature (e.g. histology). This work paves the way for using PC-CT in a wide range of TE applications which can be pivotal for unlocking the potential of this field.

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.

Dynamic Multicontrast X-Ray Imaging Method Applied to Additive Manufacturing.

We present a dynamic implementation of the beam-tracking x-ray imaging method providing absorption, phase, and ultrasmall angle scattering signals with microscopic resolution and high frame rate. We demonstrate the method's ability to capture dynamic processes with 22-ms time resolution by investigating the melting of metals in laser additive manufacturing, which has so far been limited to single-modality synchrotron radiography. The simultaneous availability of three contrast channels enables earlier segmentation of droplets, tracking of powder dynamic, and estimation of unfused powder amounts, demonstrating that the method can provide additional information on melting processes.

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.

Edge-illumination x-ray phase-contrast imaging.

Although early demonstration dates back to the mid-sixties, x-ray phase-contrast imaging (XPCI) became hugely popular in the mid-90s, thanks to the advent of 3rd generation synchrotron facilities. Its ability to reveal object features that had so far been considered invisible to x-rays immediately suggested great potential for applications across the life and the physical sciences, and an increasing number of groups worldwide started experimenting with it. At that time, it looked like a synchrotron facility was strictly necessary to perform XPCI with some degree of efficiency-the only alternative being micro-focal sources, the limited flux of which imposed excessively long exposure times. However, new approaches emerged in the mid-00s that overcame this limitation, and allowed XPCI implementations with conventional, non-micro-focal x-ray sources. One of these approaches showing particular promise for 'real-world' applications is edge-illumination XPCI: this article describes the key steps in its evolution in the context of contemporary developments in XPCI research, and presents its current state-of-the-art, especially in terms of transition towards practical applications.

Bus crash risk evaluation: An adjusted framework and its application in a real network.

Greater attention to bus safety can lead to relevant benefits for public transport companies in terms of higher service performance, reliability, and lower insurance costs. Therefore, measuring the crash risk on bus routes provides an opportunity to improve the safety performance of transit operators. Previous research has explored the effects of many factors regarding the frequency and severity of bus crashes, whereas only a handful of studies have defined some crash risk indexes. Conversely, to the best of our knowledge, almost no research has been done regarding the crash risk in the bus transit network that integrates frequency, severity, and the exposure factors. This paper proposes a new framework to assess the crash risk for each transit bus route by the integration of safety factors, prediction models and risk methods. More precisely, this framework identifies several safety factors and specifies the risk components in terms of frequency, severity and exposure factors that may affect bus crashes. Then, it models their relationships to build a bus crash risk function. Lastly, according to the values returned by the previous function, the crash risk for each route is computed and a safety performance ranking for each route is provided. The feasibility of this framework is demonstrated in a real case study by using bus crash data provided by a mid-sized Italian bus operator. The findings show that transit managers could implement this framework in a road traffic safety management system to evaluate the risk of crashes on routes, monitor the safety performance of each route and qualify each route according to recent safety norms.

Technical Note: Practical implementation strategies of cycloidal computed tomography.

PURPOSE: Cycloidal computed tomography is a novel imaging concept which combines a highly structured x-ray beam, offset lateral under-sampling, and mathematical data recovery to obtain high-resolution images efficiently and flexibly, even with relatively large source focal spots and detector pixels. The method reduces scanning time and, potentially, delivered dose compared to other sampling schemes. This study aims to present and discuss several implementation strategies for cycloidal computed tomography (CT) in order to increase its ease of use and facilitate uptake within the imaging community. METHODS: The different implementation strategies presented are step-and-shoot, continuous unidirectional, continuous back-and-forth, and continuous pixel-wise scanning. In step-and-shoot scans the sample remains stationary while frames are acquired, whereas in all other cases the sample moves through the scanner continuously. The difference between the continuous approaches is the trajectory by which the sample moves within the field of view. RESULTS: All four implementation strategies are compatible with a standard table-top x-ray setup. With the experimental setup applied here, step-and-shoot acquisitions yield the best spatial resolution (around 30 µm), but are the most time-consuming (1.4 h). Continuous unidirectional and back-and-forth images have resolution between 30 and 40 µm, and are faster (35 min). Continuous pixel-wise images are equally time-efficient, although technical challenges caused a small loss in image quality with a resolution of about 50 µm. CONCLUSION: The authors show that cycloidal CT can be implemented in a variety of ways with high quality results. They believe this posits cycloidal CT as a powerful imaging alternative to more time-consuming and less flexible methods in the field.