Dual energy X-ray beam ptycho-fluorescence imaging.

X-ray ptychography and X-ray fluorescence are complementary nanoscale imaging techniques, providing structural and elemental information, respectively. Both methods acquire data by scanning a localized beam across the sample. X-ray ptychography processes the transmission signal of a coherent illumination interacting with the sample, to produce images with a resolution finer than the illumination spot and step size. By enlarging both the spot and the step size, the technique can cover extended regions efficiently. X-ray fluorescence records the emitted spectra as the sample is scanned through the localized beam and its spatial resolution is limited by the spot and step size. The requisites for fast ptychography and high-resolution fluorescence appear incompatible. Here, a novel scheme that mitigates the difference in requirements is proposed. The method makes use of two probes of different sizes at the sample, generated by using two different energies for the probes and chromatic focusing optics. The different probe sizes allow to reduce the number of acquisition steps for the joint fluorescence-ptychography scan compared with a standard single beam scan, while imaging the same field of view. The new method is demonstrated experimentally using two undulator harmonics, a Fresnel zone plate and an energy discriminating photon counting detector.

Exploring Alzheimer's disease mouse brain through X-ray phase contrast tomography: From the cell to the organ.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aß) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aß plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.

Post-reconstruction 3D single-distance phase retrieval for multi-stage phase-contrast tomography with photon-counting detectors.

In the case of single-distance propagation-based phase-contrast X-ray computed tomography with synchrotron radiation, the conventional reconstruction pipeline includes an independent 2D phase retrieval filtering of each acquired projection prior to the actual reconstruction. In order to compensate for the limited height of the X-ray beam or the small sensitive area of most modern X-ray photon-counting detectors, it is quite common to image large objects with a multi-stage approach, i.e. several acquisitions at different vertical positions of the sample. In this context, the conventional reconstruction pipeline may introduce artifacts at the margins of each vertical stage. This article presents a modified computational protocol where a post-reconstruction 3D volume phase retrieval is applied. By comparing the conventional 2D and the proposed 3D reconstructions of a large mastectomy specimen (9 cm in diameter and 3 cm in height), it is here shown that the 3D approach compensates for the multi-stage artifacts, it avoids refined projection stitching, and the image quality in terms of spatial resolution, contrast and contrast-to-noise ratio is preserved.

Large-area single-photon-counting CdTe detector for synchrotron radiation computed tomography: a dedicated pre-processing procedure.

Large-area CdTe single-photon-counting detectors are becoming more and more attractive in view of low-dose imaging applications due to their high efficiency, low intrinsic noise and absence of a scintillating screen which affects spatial resolution. At present, however, since the dimensions of a single sensor are small (typically a few cm2), multi-module architectures are needed to obtain a large field of view. This requires coping with inter-module gaps and with close-to-edge pixels, which generally show a non-optimal behavior. Moreover, high-Z detectors often show gain variations in time due to charge trapping: this effect is detrimental especially in computed tomography (CT) applications where a single tomographic image requires hundreds of projections continuously acquired in several seconds. This work has been carried out at the SYRMEP beamline of the Elettra synchrotron radiation facility (Trieste, Italy), in the framework of the SYRMA-3D project, which aims to perform the world's first breast-CT clinical study with synchrotron radiation. An ad hoc data pre-processing procedure has been developed for the PIXIRAD-8 CdTe single-photon-counting detector, comprising an array of eight 30.7 mm × 24.8 mm modules tiling a 246 mm × 25 mm sensitive area, which covers the full synchrotron radiation beam. The procedure consists of five building blocks, namely dynamic flat-fielding, gap seaming, dynamic ring removal, projection despeckling and around-gap equalization. Each block is discussed and compared, when existing, with conventional approaches. The effectiveness of the pre-processing is demonstrated for phase-contrast CT images of a human breast specimen. The dynamic nature of the proposed procedure, which provides corrections dependent upon the projection index, allows the effective removal of time-dependent artifacts, preserving the main image features including phase effects.

Integrating longitudinal information in hippocampal volume measurements for the early detection of Alzheimer's disease.

BACKGROUND: Structural MRI measures for monitoring Alzheimer's Disease (AD) progression are becoming instrumental in the clinical practice, and more so in the context of longitudinal studies. This investigation addresses the impact of four image analysis approaches on the longitudinal performance of the hippocampal volume. METHODS: We present a hippocampal segmentation algorithm and validate it on a gold-standard manual tracing database. We segmented 460 subjects from ADNI, each subject having been scanned twice at baseline, 12-month and 24month follow-up scan (1.5T, T1 MRI). We used the bilateral hippocampal volume v and its variation, measured as the annualized volume change Λ=δv/year(mm(3)/y). Four processing approaches with different complexity are compared to maximize the longitudinal information, and they are tested for cohort discrimination ability. Reference cohorts are Controls vs. Alzheimer's Disease (CTRL/AD) and CTRL vs. Mild Cognitive Impairment who subsequently progressed to AD dementia (CTRL/MCI-co). We discuss the conditions on v and the added value of Λ in discriminating subjects. RESULTS: The age-corrected bilateral annualized atrophy rate (%/year) were: -1.6 (0.6) for CTRL, -2.2 (1.0) for MCI-nc, -3.2 (1.2) for MCI-co and -4.0 (1.5) for AD. Combined (v, Λ) discrimination ability gave an Area under the ROC curve (auc)=0.93 for CTRL vs AD and auc=0.88 for CTRL vs MCI-co. CONCLUSIONS: Longitudinal volume measurements can provide meaningful clinical insight and added value with respect to the baseline provided the analysis procedure embeds the longitudinal information.

A feasibility study of X-ray phase-contrast mammographic tomography at the Imaging and Medical beamline of the Australian Synchrotron.

Results are presented of a recent experiment at the Imaging and Medical beamline of the Australian Synchrotron intended to contribute to the implementation of low-dose high-sensitivity three-dimensional mammographic phase-contrast imaging, initially at synchrotrons and subsequently in hospitals and medical imaging clinics. The effect of such imaging parameters as X-ray energy, source size, detector resolution, sample-to-detector distance, scanning and data processing strategies in the case of propagation-based phase-contrast computed tomography (CT) have been tested, quantified, evaluated and optimized using a plastic phantom simulating relevant breast-tissue characteristics. Analysis of the data collected using a Hamamatsu CMOS Flat Panel Sensor, with a pixel size of 100 µm, revealed the presence of propagation-based phase contrast and demonstrated significant improvement of the quality of phase-contrast CT imaging compared with conventional (absorption-based) CT, at medically acceptable radiation doses.

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.

PEPI Lab: a flexible compact multi-modal setup for X-ray phase-contrast and spectral imaging.

This paper presents a new flexible compact multi-modal imaging setup referred to as PEPI (Photon-counting Edge-illumination Phase-contrast imaging) Lab, which is based on the edge-illumination (EI) technique and a chromatic detector. The system enables both X-ray phase-contrast (XPCI) and spectral (XSI) imaging of samples on the centimeter scale. This work conceptually follows all the stages in its realization, from the design to the first imaging results. The setup can be operated in four different modes, i.e. photon-counting/conventional, spectral, double-mask EI, and single-mask EI, whereby the switch to any modality is fast, software controlled, and does not require any hardware modification or lengthy re-alignment procedures. The system specifications, ranging from the X-ray tube features to the mask material and aspect ratio, have been quantitatively studied and optimized through a dedicated Geant4 simulation platform, guiding the choice of the instrumentation. The realization of the imaging setup, both in terms of hardware and control software, is detailed and discussed with a focus on practical/experimental aspects. Flexibility and compactness (66 cm source-to-detector distance in EI) are ensured by dedicated motion stages, whereas spectral capabilities are enabled by the Pixirad-1/Pixie-III detector in combination with a tungsten anode X-ray source operating in the range 40-100 kVp. The stability of the system, when operated in EI, has been verified, and drifts leading to mask misalignment of less than 1 [Formula: see text]m have been measured over a period of 54 h. The first imaging results, one for each modality, demonstrate that the system fulfills its design requirements. Specifically, XSI tomographic images of an iodine-based phantom demonstrate the system's quantitativeness and sensibility to concentrations in the order of a few mg/ml. Planar XPCI images of a carpenter bee specimen, both in single and double-mask modes, demonstrate that refraction sensitivity (below 0.6 [Formula: see text]rad in double-mask mode) is comparable with other XPCI systems based on microfocus sources. Phase CT capabilities have also been tested on a dedicated plastic phantom, where the phase channel yielded a 15-fold higher signal-to-noise ratio with respect to attenuation.

Lesion Extension and Neuronal Loss after Spinal Cord Injury Using X-Ray Phase-Contrast Tomography in Mice.

Following spinal cord injury (SCI) the degree of functional (motor, autonomous, or sensory) loss correlates with the severity of nervous tissue damage. An imaging technique able to capture non-invasively and simultaneously the complex mechanisms of neuronal loss, vascular damage, and peri-lesional tissue reorganization is currently lacking in experimental SCI studies. Synchrotron X-ray phase-contrast tomography (SXPCT) has emerged as a non-destructive three-dimensional (3D) neuroimaging technique with high contrast and spatial resolution. In this framework, we developed a multi-modal approach combining SXPCT, histology and correlative methods to study neurovascular architecture in normal and spinal level C4-contused mouse spinal cords (C57BL/6J mice, age 2-3 months). The evolution of SCI lesion was imaged at the cell resolution level during the acute (30 min) and subacute (7 day) phases. Spared motor neurons (MNs) were segmented and quantified in different volumes localized at and away from the epicenter. SXPCT was able to capture neuronal loss and blood-brain barrier breakdown following SCI. Three-dimensional quantification based on SXPCT acquisitions showed no additional MN loss between 30 min and 7 days post-SCI. In addition, the analysis of hemorrhagic (at 30 min) and lesion (at 7 days) volumes revealed a high similarity in size, suggesting no extension of tissue degeneration between early and later time-points. Moreover, glial scar borders were unevenly distributed, with rostral edges being the most extended. In conclusion, SXPCT capability to image at high resolution cellular changes in 3D enables the understanding of the relationship between hemorrhagic events and nervous structure damage in SCI.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

PyPore3D: An Open Source Software Tool for Imaging Data Processing and Analysis of Porous and Multiphase Media.

In this work, we propose the software library PyPore3D, an open source solution for data processing of large 3D/4D tomographic data sets. PyPore3D is based on the Pore3D core library, developed thanks to the collaboration between Elettra Sincrotrone (Trieste) and the University of Trieste (Italy). The Pore3D core library is built with a distinction between the User Interface and the backend filtering, segmentation, morphological processing, skeletonisation and analysis functions. The current Pore3D version relies on the closed source IDL framework to call the backend functions and enables simple scripting procedures for streamlined data processing. PyPore3D addresses this limitation by proposing a full open source solution which provides Python wrappers to the the Pore3D C library functions. The PyPore3D library allows the users to fully use the Pore3D Core Library as an open source solution under Python and Jupyter Notebooks PyPore3D is both getting rid of all the intrinsic limitations of licensed platforms (e.g., closed source and export restrictions) and adding, when needed, the flexibility of being able to integrate scientific libraries available for Python (SciPy, TensorFlow, etc.).

Investigation of the microstructure and mineralogical composition of urinary calculi fragments by synchrotron radiation X-ray microtomography: a feasibility study.

The outcomes from the feasibility study on utilization of synchrotron radiation X-ray microtomography (SR-µCT) to investigate the texture and the quantitative mineralogical composition of selected calcium oxalate-based urinary calculi fragments are presented. The comparison of the results obtained by SR-µCT analysis with those derived from current standard analytical approaches is provided. SR-µCT is proved as a potential effective technique for determination of texture, 3D microstructure, and composition of kidney stones.

Automated quantitative characterization of alginate/hydroxyapatite bone tissue engineering scaffolds by means of micro-CT image analysis.

Accurate image acquisition techniques and analysis protocols for a reliable characterization of tissue engineering scaffolds are yet to be well defined. To this aim, the most promising imaging technique seems to be the X-ray computed microtomography (µ-CT). However critical issues of the analysis process deal with the representativeness of the selected Volume of Interest (VOI) and, most significantly, its segmentation. This article presents an image analysis protocol that computes a set of quantitative descriptors suitable for characterizing the morphology and the micro-architecture of alginate/hydroxyapatite bone tissue engineering scaffolds. Considering different VOIs extracted from different µ-CT datasets, an automated segmentation technique is suggested and compared against a manual segmentation. Variable sizes of VOIs are also considered in order to assess their representativeness. The resulting image analysis protocol is reproducible, parameter-free and it automatically provides accurate quantitative information in addition to the simple qualitative observation of the acquired images.

Asbestos bodies count and morphometry in bulk lung tissue samples by non-invasive X-ray micro-tomography.

The number of the Asbestos Bodies (AB), i.e. asbestos that developed an iron-protein coating during its permanence in biological tissues, is one of the most accessible markers of asbestos exposure in individuals. The approaches developed to perform AB count in biological tissues are based on the manual examination of tissue digests or histological sections by means of light or electron microscopies. Although these approaches are well established and relatively accessible, manual examination is time-consuming and can be reader-dependent. Besides, approximations are applied because of the limitations of 2D readings and to speed up manual counts. In addition, sample preparation using tissue digests require an amount of tissue that can only be obtained by invasive surgery or post-mortem sampling. In this paper, we propose a new approach to AB counting based on non-destructive 3D imaging, which has the potential to overcome most of the limitations of conventional approaches. This method allows automating the AB count and determining their morphometry distribution in bulk tissue samples (ideally non-invasive needle biopsies), with minimal sample preparation and avoiding approximations. Although the results are promising, additional testing on a larger number of AB-containing biological samples would be required to fully validate the method.

Chemo-physical properties of asbestos bodies in human lung tissues studied at the nano-scale by non-invasive, label free x-ray imaging and spectroscopic techniques.

In the lungs, asbestos develops an Fe-rich coating (Asbestos Body, AB) that becomes the actual interface between the foreign fibers and the host organism. Conventional approaches to study ABs require an invasive sample preparation that can alter them. In this work, a novel combination of x-ray tomography and spectroscopy allowed studying unaltered lung tissue samples with chrysotile and crocidolite asbestos. The thickness and mass density maps of the ABs obtained by x-ray tomography were used to derive a truly quantitative elemental analysis from scanning x-ray fluorescence spectroscopy data. The average mass density of the ABs is compatible with that of highly loaded ferritin, or hemosiderin. The composition of all ABs analyzed was similar, with only minor differences in the relative elemental fractions. Silicon concentration decreased in the core-to-rim direction, indicating a possible partial dissolution of the inner fiber. The Fe content in the ABs was higher than that possibly contained in chrysotile and crocidolite. This finding opens two opposite scenarios, the first with Fe coming from the fiber bulk and concentrating on the surface as long as the fiber dissolves, the second where the Fe that takes part to the formation of the AB originates from the host organism Fe-pool.

Motion artifacts assessment and correction using optical tracking in synchrotron radiation breast CT.

PURPOSE: The SYRMA-3D collaboration is setting up a breast computed tomography (bCT) clinical program at the Elettra synchrotron radiation facility in Trieste, Italy. Unlike the few dedicated scanners available at hospitals, synchrotron radiation bCT requires the patient's rotation, which in turn implies a long scan duration (from tens of seconds to few minutes). At the same time, it allows the achievement of high spatial resolution. These features make synchrotron radiation bCT prone to motion artifacts. This article aims at assessing and compensating for motion artifacts through an optical tracking approach. METHODS: In this study, patients' movements due to breathing have been first assessed on seven volunteers and then simulated during the CT scans of a breast phantom and a surgical specimen, by adding a periodic oscillatory motion (constant speed, 1 mm amplitude, 12 cycles/minute). CT scans were carried out at 28 keV with a mean glandular dose of 5 mGy. Motion artifacts were evaluated and a correction algorithm based on the optical tracking of fiducial marks was introduced. A quantitative analysis based on the structural similarity (SSIM) index and the normalized mean square error (nMSE) was performed on the reconstructed CT images. RESULTS: CT images reconstructed through the optical tracking procedure were found to be as good as the motionless reference image. Moreover, the analysis of SSIM and nMSE demonstrated that an uncorrected motion of the order of the system's point spread function (around 0.1 mm in the present case) can be tolerated. CONCLUSIONS: Results suggest that a motion correction procedure based on an optical tracking system would be beneficial in synchrotron radiation bCT.

3D Spatial Distribution of Nanoparticles in Mice Brain Metastases by X-ray Phase-Contrast Tomography.

Characterizing nanoparticles (NPs) distribution in multiple and complex metastases is of fundamental relevance for the development of radiological protocols based on NPs administration. In the literature, there have been advances in monitoring NPs in tissues. However, the lack of 3D information is still an issue. X-ray phase-contrast tomography (XPCT) is a 3D label-free, non-invasive and multi-scale approach allowing imaging anatomical details with high spatial and contrast resolutions. Here an XPCT qualitative study on NPs distribution in a mouse brain model of melanoma metastases injected with gadolinium-based NPs for theranostics is presented. For the first time, XPCT images show the NPs uptake at micrometer resolution over the full brain. Our results revealed a heterogeneous distribution of the NPs inside the melanoma metastases, bridging the gap in spatial resolution between magnetic resonance imaging and histology. Our findings demonstrated that XPCT is a reliable technique for NPs detection and can be considered as an emerging method for the study of NPs distribution in organs.

Edge-subtraction X-ray ptychographic imaging with pink beam synchrotron radiation and a single photon-counting detector.

We present here a new method of performing X-ray edge-subtraction ptychographic imaging by combining multiple harmonics from an undulator synchtrotron source and an energy discriminating photon counting detector. Conventionally, monochromatic far-field X-ray ptychography is used to perform edge subtraction through the use of multiple monochromatic energy scans to obtain spectral information for a variety of applications. Here, we use directly the undulator spectrum from a synchrotron source, selecting two separate harmonics post sample using the Pixirad-1/Pixie-III detector. The result is two monochromatic images, above and below an absorption edge of interest. The proposed method is applied to obtain Au L-edge subtraction imaging of a Au-Ni grid test sample. The Au L-edge subtraction is particularly relevant for the identification of gold nanoparticles for biomedical applications. Switching the energy scan mechanism from a mechanical monochromator to an electronic detector threshold allows for faster spectral data collection with improved stability.

High resolution 3D visualization of the spinal cord in a post-mortem murine model.

A crucial issue in the development of therapies to treat pathologies of the central nervous system is represented by the availability of non-invasive methods to study the three-dimensional morphology of spinal cord, with a resolution able to characterize its complex vascular and neuronal organization. X-ray phase contrast micro-tomography enables a high-quality, 3D visualization of both the vascular and neuronal network simultaneously without the need of contrast agents, destructive sample preparations or sectioning. Until now, high resolution investigations of the post-mortem spinal cord in murine models have mostly been performed in spinal cords removed from the spinal canal. We present here post-mortem phase contrast micro-tomography images reconstructed using advanced computational tools to obtain high-resolution and high-contrast 3D images of the fixed spinal cord without removing the bones and preserving the richness of micro-details available when measuring exposed spinal cords. We believe that it represents a significant step toward the in-vivo application.

Assessment of plaque morphology in Alzheimer's mouse cerebellum using three-dimensional X-ray phase-based virtual histology.

Visualization and characterization of [Formula: see text]-amyloid deposits is a fundamental task in pre-clinical study of Alzheimer's disease (AD) to assess its evolution and monitor the efficiency of new therapeutic strategies. While the cerebellum is one of the brain areas most underestimated in the context of AD, renewed interest in cerebellar lesions has recently arisen as they may link to motor and cognitive alterations. Thus, we quantitatively investigated three-dimensional plaque morphology in the cerebellum in APP/PS1 transgenic mouse, as a model of AD. In order to obtain a complete high-resolution three-dimensional view of the investigated tissue, we exploited synchrotron X-ray phase contrast tomography (XPCT), providing virtual slices with histology-matching resolution. We found the formation of plaques elongated in shape, and with a specific orientation in space depending on the investigated region of the cerebellar cortex. Remarkably, a similar shape is observed in human cerebellum from demented patients. Our findings demonstrate the capability of XPCT in volumetric quantification, supporting the current knowledge about plaque morphology in the cerebellum and the fundamental role of the surrounding tissue in driving their evolution. A good correlation with the human neuropathology is also reported.