Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses are not affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

A Novel Three-Dimensional Approach Towards Evaluating Endomyocardial Biopsies for Follow-Up After Heart Transplantation: X-Ray Phase Contrast Imaging and Its Agreement With Classical Histopathology.

Endomyocardial biopsies are the gold standard for surveillance of graft rejection following heart transplantation, and are assessed by classical histopathology using a limited number of previously stained slices from several biopsies. Synchrotron propagation-based X-ray phase contrast imaging is a non-destructive method to image biological samples without tissue preparation, enabling virtual 2D and 3D histopathology. We aimed to show the feasibility of this method to assess acute cellular rejection and its agreement to classical histopathology. Right ventricular biopsies were sampled from 23 heart transplantation recipients (20 males, mean age 54±14 years) as part of standard follow-up. The clinical diagnosis of potential rejection was made using classical histopathology. One additional study sample was harvested and imaged by X-ray phase contrast imaging, producing 3D datasets with 0.65 µm pixel size, and up to 4,320 images per sample. An experienced pathologist graded both histopathological and X-ray phase contrast images in a blinded fashion. The agreement between methods was assessed by weighted kappa, showing substantial agreement (kappa up to 0.80, p < 0.01) between X-ray phase contrast imaging and classical histopathology. X-ray phase contrast imaging does not require tissue processing, allows thorough analysis of a full myocardial sample and allows identification of acute cellular rejection.

Multimodal Image Fusion for X-ray Grating Interferometry.

X-ray grating interferometry (XGI) can provide multiple image modalities. It does so by utilizing three different contrast mechanisms-attenuation, refraction (differential phase-shift), and scattering (dark-field)-in a single dataset. Combining all three imaging modalities could create new opportunities for the characterization of material structure features that conventional attenuation-based methods are unable probe. In this study, we proposed an image fusion scheme based on the non-subsampled contourlet transform and spiking cortical model (NSCT-SCM) to combine the tri-contrast images retrieved from XGI. It incorporated three main steps: (i) image denoising based on Wiener filtering, (ii) the NSCT-SCM tri-contrast fusion algorithm, and (iii) image enhancement using contrast-limited adaptive histogram equalization, adaptive sharpening, and gamma correction. The tri-contrast images of the frog toes were used to validate the proposed approach. Moreover, the proposed method was compared with three other image fusion methods by several figures of merit. The experimental evaluation results highlighted the efficiency and robustness of the proposed scheme, with less noise, higher contrast, more information, and better details.

A novel fusion method for X-ray phase contrast imaging based on fast adaptive bidimensional empirical mode decomposition.

BACKGROUNDS: X-ray phase contrast imaging (XPCI) can separate the attenuation, refraction, and scattering signals of the object. The application of image fusion enables the concentration of distinctive information into a single image. Some methods have been applied in XPCI field, but wavelet-based decomposition approaches often result in loss of original data. OBJECTIVE: To explore the application value of a novel image fusion method for XPCI system and computed tomography (CT) system. METHODS: The means of fast adaptive bidimensional empirical mode decomposition (FABEMD) is considered for image decomposition to avoid unnecessary information loss. A parameter δ is proposed to guide the fusion of bidimensional intrinsic mode functions which contain high-frequency information, using a pulse coupled neural network with morphological gradients (MGPCNN). The residual images are fused by the energy attribute fusion strategy. Image preprocessing and enhancement are performed on the result to ensure its quality. The effectiveness of other image fusion methods has been compared, such as discrete wavelet transforms and anisotropic diffusion fusion. RESULTS: The δ-guided FABEMD-MGPCNN method achieved either the first or second position in objective evaluation metrics with biological samples, as compared to other image fusion methods. Moreover, comparisons are made with other fusion methods used for XPCI. Finally, the proposed method applied in CT show expected results to retain the feature information. CONCLUSIONS: The proposed δ-guided FABEMD-MGPCNN method shows potential feasibility and superiority over traditional and recent image fusion methods for X-ray differential phase contrast imaging and computed tomography systems.

Direct Conversion X-ray Detector with Micron-Scale Pixel Pitch for Edge-Illumination and Propagation-Based X-ray Phase-Contrast Imaging.

In this work, we investigate the potential of employing a direct conversion integration mode X-ray detector with micron-scale pixels in two different X-ray phase-contrast imaging (XPCi) configurations, propagation-based (PB) and edge illumination (EI). Both PB-XPCi and EI-XPCi implementations are evaluated through a wave optics model-numerically simulated in MATLAB-and are compared based on their contrast, edge-enhancement, visibility, and dose efficiency characteristics. The EI-XPCi configuration, in general, demonstrates higher performance compared to PB-XPCi, considering a setup with the same X-ray source and detector. However, absorption masks quality (thickness of X-ray absorption material) and environmental vibration effect are two potential challenges for EI-XPCi employing a detector with micron-scale pixels. Simulation results confirm that the behavior of an EI-XPCi system employing a high-resolution detector is susceptible to its absorption masks thickness and misalignment. This work demonstrates the potential and feasibility of employing a high-resolution direct conversion detector for phase-contrast imaging applications where higher dose efficiency, higher contrast images, and a more compact imaging system are of interest.

Multiscale X-ray phase contrast imaging of human cartilage for investigating osteoarthritis formation.

BACKGROUND: The evolution of cartilage degeneration is still not fully understood, partly due to its thinness, low radio-opacity and therefore lack of adequately resolving imaging techniques. X-ray phase-contrast imaging (X-PCI) offers increased sensitivity with respect to standard radiography and CT allowing an enhanced visibility of adjoining, low density structures with an almost histological image resolution. This study examined the feasibility of X-PCI for high-resolution (sub-) micrometer analysis of different stages in tissue degeneration of human cartilage samples and compare it to histology and transmission electron microscopy. METHODS: Ten 10%-formalin preserved healthy and moderately degenerated osteochondral samples, post-mortem extracted from human knee joints, were examined using four different X-PCI tomographic set-ups using synchrotron radiation the European Synchrotron Radiation Facility (France) and the Swiss Light Source (Switzerland). Volumetric datasets were acquired with voxel sizes between 0.7 × 0.7 × 0.7 and 0.1 × 0.1 × 0.1 µm3. Data were reconstructed by a filtered back-projection algorithm, post-processed by ImageJ, the WEKA machine learning pixel classification tool and VGStudio max. For correlation, osteochondral samples were processed for histology and transmission electron microscopy. RESULTS: X-PCI provides a three-dimensional visualization of healthy and moderately degenerated cartilage samples down to a (sub-)cellular level with good correlation to histologic and transmission electron microscopy images. X-PCI is able to resolve the three layers and the architectural organization of cartilage including changes in chondrocyte cell morphology, chondrocyte subgroup distribution and (re-)organization as well as its subtle matrix structures. CONCLUSIONS: X-PCI captures comprehensive cartilage tissue transformation in its environment and might serve as a tissue-preserving, staining-free and volumetric virtual histology tool for examining and chronicling cartilage behavior in basic research/laboratory experiments of cartilage disease evolution.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

The versatile X-ray beamline of the Munich Compact Light Source: design, instrumentation and applications.

Inverse Compton scattering provides means to generate low-divergence partially coherent quasi-monochromatic, i.e. synchrotron-like, X-ray radiation on a laboratory scale. This enables the transfer of synchrotron techniques into university or industrial environments. Here, the Munich Compact Light Source is presented, which is such a compact synchrotron radiation facility based on an inverse Compton X-ray source (ICS). The recent improvements of the ICS are reported first and then the various experimental techniques which are most suited to the ICS installed at the Technical University of Munich are reviewed. For the latter, a multipurpose X-ray application beamline with two end-stations was designed. The beamline's design and geometry are presented in detail including the different set-ups as well as the available detector options. Application examples of the classes of experiments that can be performed are summarized afterwards. Among them are dynamic in vivo respiratory imaging, propagation-based phase-contrast imaging, grating-based phase-contrast imaging, X-ray microtomography, K-edge subtraction imaging and X-ray spectroscopy. Finally, plans to upgrade the beamline in order to enhance its capabilities are discussed.

Showcasing the application of synchrotron-based X-ray computed tomography in host-pathogen interactions: The role of wheat rachilla and rachis nodes in Type-II resistance to Fusarium graminearum.

Fusarium head blight, caused primarily by Fusarium graminearum (Fg), is one of the most devastating diseases of wheat. Host resistance in wheat is classified into five types (Type-I to Type-V), and a majority of moderately resistant genotypes carry Type-II resistance (resistance to pathogen spread in the rachis) alleles, mainly from the Chinese cultivar Sumai 3. Histopathological studies in the past failed to identify the key tissue in the spike conferring resistance to pathogen spread, and most of the studies used destructive techniques, potentially damaging the tissue(s) under study. In the present study, nondestructive synchrotron-based phase contrast X-ray imaging and computed tomography techniques were used to confirm the part of the wheat spike conferring Type-II resistance to Fg spread, thus showcasing the application of synchrotron-based techniques to image host-pathogen interactions. Seven wheat genotypes of moderate resistance to Fusarium head blight were studied for changes in the void space volume fraction and grayscale/voxel intensity following Fg inoculation. Cell-wall biopolymeric compounds were quantified using Fourier-transform midinfrared spectroscopy for all genotype-treatment combinations. The study revealed that the rachilla and rachis nodes together are structurally important in conferring Type-II resistance. The structural reinforcement was not necessarily observed from lignin deposition but rather from an unknown mechanism.

The optical stretcher as a tool for single-particle X-ray imaging and diffraction.

For almost half a century, optical tweezers have successfully been used to micromanipulate micrometre and sub-micrometre-sized particles. However, in recent years it has been shown experimentally that, compared with single-beam traps, the use of two opposing and divergent laser beams can be more suitable in studying the elastic properties of biological cells and vesicles. Such a configuration is termed an optical stretcher due to its capability of applying high deforming forces on biological objects such as cells. In this article the experimental capabilities of an optical stretcher as a potential sample delivery system for X-ray diffraction and imaging studies at synchrotrons and X-ray free-electron laser (FEL) facilites are demonstrated. To highlight the potential of the optical stretcher its micromanipulation capabilities have been used to image polymer beads and label biological cells. Even in a non-optimized configuration based on a commercially available optical stretcher system, X-ray holograms could be recorded from different views on a biological cell and the three-dimensional phase of the cell could be reconstructed. The capability of the setup to deform cells at higher laser intensities in combination with, for example, X-ray diffraction studies could furthermore lead to interesting studies that couple structural parameters to elastic properties. By means of high-throughput screening, the optical stretcher could become a useful tool in X-ray studies employing synchrotron radiation, and, at a later stage, femtosecond X-ray pulses delivered by X-ray free-electron lasers.

X-ray analyzer-based phase-contrast computed laminography.

X-ray analyzer-based phase-contrast imaging is combined with computed laminography for imaging regions of interest in laterally extended flat specimens of weak absorption contrast. The optics discussed here consist of an asymmetrically cut collimator crystal and a symmetrically cut analyzer crystal arranged in a nondispersive (+, -) diffraction geometry. A generalized algorithm is given for calculating multi-contrast (absorption, refraction and phase contrast) images of a sample. Basic formulae are also presented for laminographic reconstruction. The feasibility of the method discussed was verified at the vertical wiggler beamline BL-14B of the Photon Factory. At a wavelength of 0.0733 nm, phase-contrast sectional images of plastic beads were successfully obtained. Owing to strong circular artifacts caused by a sample holder, the field of view was limited to about 6 mm in diameter.

Increasing the field of view in grating based X-ray phase contrast imaging using stitched gratings.

Grating based X-ray differential phase contrast imaging (DPCI) allows for high contrast imaging of materials with similar absorption characteristics. In the last years' publications, small animals or parts of the human body like breast, hand, joints or blood vessels have been studied. Larger objects could not be investigated due to the restricted field of view limited by the available grating area. In this paper, we report on a new stitching method to increase the grating area significantly: individual gratings are merged on a carrier substrate. Whereas the grating fabrication process is based on the LIGA technology (X-ray lithography and electroplating) different cutting and joining methods have been evaluated. First imaging results using a 2×2 stitched analyzer grating in a Talbot-Lau interferometer have been generated using a conventional polychromatic X-ray source. The image quality and analysis confirm the high potential of the stitching method to increase the field of view considerably.

A novel imaging tool for hepatic portal system using phase contrast technique with hydrogen peroxide-generated O2 gas.

The objective of this study was to investigate the potential of hydrogen peroxide-generated oxygen gas-based phase contrast imaging (PCI) for visualizing mouse hepatic portal veins. The O2 gas was made from the reaction between H2O2 and catalase. The gas production was imaged by PCI in real time. The H2O2 was injected into the enteric cavity of the lower sigmoid colon to produce O2 in the submucosal venous plexus. The generated O2 gas could be finally drained into hepatic portal veins. Absorption contrast imaging (ACI) and PCI of O2-filled portal veins were performed and compared. PCI offers high resolution and real-time visualization of the O2 gas production. Compared with O2-based ACI, O2-based PCI significantly enhanced the revealing of the portal vein in vivo. It is concluded that O2-based PCI is a novel and promising imaging modality for future studies of portal venous disorders in mice models.

Visualization of water drying in porous materials by X-ray phase contrast imaging.

We present in this study results from X-ray tomographic microscopy with synchrotron radiation performed both in attenuation and phase contrast modes on a limestone sample during two stages of water drying. No contrast agent was used in order to increase the X-ray attenuation by water. We show that only by using the phase contrast mode it is possible to achieve enough water content change resolution to investigate the drying process at the pore-scale. We performed 3D image analysis of the time-differential phase contrast tomogram. We show by the results of such analysis that it is possible to obtain a reliable characterization of the spatial redistribution of water in the resolved pore system in agreement with what expected from the theory of drying in porous media and from measurements performed with other approaches. We thus show the potential of X-ray phase contrast imaging for pore-scale investigations of reactive water transport processes which cannot be imaged by adding a contrast agent for exploiting the standard attenuation contrast imaging mode.

Improved visualization of breast cancer features in multifocal carcinoma using phase-contrast and dark-field mammography: an ex vivo study.

OBJECTIVES: Conventional X-ray attenuation-based contrast is inherently low for the soft-tissue components of the female breast. To overcome this limitation, we investigate the diagnostic merits arising from dark-field mammography by means of certain tumour structures enclosed within freshly dissected mastectomy samples. METHODS: We performed grating-based absorption, absolute phase and dark-field mammography of three freshly dissected mastectomy samples containing bi- and multifocal carcinoma using a compact, laboratory Talbot-Lau interferometer. Preoperative in vivo imaging (digital mammography, ultrasound, MRI), postoperative histopathological analysis and ex vivo digital mammograms of all samples were acquired for the diagnostic verification of our results. RESULTS: In the diagnosis of multifocal tumour growth, dark-field mammography seems superior to standard breast imaging modalities, providing a better resolution of small, calcified tumour nodules, demarcation of tumour boundaries with desmoplastic stromal response and spiculated soft-tissue strands extending from an invasive ductal breast cancer. CONCLUSIONS: On the basis of selected cases, we demonstrate that dark-field mammography is capable of outperforming conventional mammographic imaging of tumour features in both calcified and non-calcified tumours. Presuming dose optimization, our results encourage further studies on larger patient cohorts to identify those patients that will benefit the most from this promising additional imaging modality. KEY POINTS: • X-ray dark-field mammography provides significantly improved visualization of tumour features • X-ray dark-field mammography is capable of outperforming conventional mammographic imaging • X-ray dark-field mammography provides imaging sensitivity towards highly dispersed calcium grains.

Combining Monte Carlo methods with coherent wave optics for the simulation of phase-sensitive X-ray imaging.

Phase-sensitive X-ray imaging shows a high sensitivity towards electron density variations, making it well suited for imaging of soft tissue matter. However, there are still open questions about the details of the image formation process. Here, a framework for numerical simulations of phase-sensitive X-ray imaging is presented, which takes both particle- and wave-like properties of X-rays into consideration. A split approach is presented where we combine a Monte Carlo method (MC) based sample part with a wave optics simulation based propagation part, leading to a framework that takes both particle- and wave-like properties into account. The framework can be adapted to different phase-sensitive imaging methods and has been validated through comparisons with experiments for grating interferometry and propagation-based imaging. The validation of the framework shows that the combination of wave optics and MC has been successfully implemented and yields good agreement between measurements and simulations. This demonstrates that the physical processes relevant for developing a deeper understanding of scattering in the context of phase-sensitive imaging are modelled in a sufficiently accurate manner. The framework can be used for the simulation of phase-sensitive X-ray imaging, for instance for the simulation of grating interferometry or propagation-based imaging.

Phase retrieval for X-ray differential phase contrast radiography with knowledge transfer learning from virtual differential absorption model.

Grating-based X-ray phase contrast radiography and computed tomography (CT) are promising modalities for future medical applications. However, the ill-posed phase retrieval problem in X-ray phase contrast imaging has hindered its use for quantitative analysis in biomedical imaging. Deep learning has been proved as an effective tool for image retrieval. However, in practical grating-based X-ray phase contrast imaging system, acquiring the ground truth of phase to form image pairs is challenging, which poses a great obstacle for using deep leaning methods. Transfer learning is widely used to address the problem with knowledge inheritance from similar tasks. In the present research, we propose a virtual differential absorption model and generate a training dataset with differential absorption images and absorption images. The knowledge learned from the training is transferred to phase retrieval with transfer learning techniques. Numerical simulations and experiments both demonstrate its feasibility. Image quality of retrieved phase radiograph and phase CT slices is improved when compared with representative phase retrieval methods. We conclude that this method is helpful in both X-ray 2D and 3D imaging and may find its applications in X-ray phase contrast radiography and X-ray phase CT.

[18F]-FDG uptake in brain slices prepared from an aged mouse model of Alzheimer's disease using a dynamic autoradiography technique.

OBJECTIVE: 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography ([18F]-FDG-PET) is a imaging modality that has been used to measure of glucose metabolism in the brain in Alzheimer's disease (AD). Clinically, decreased glucose uptake has been reported in the brain of AD, although the precise underlying mechanisms have not yet been elucidated. To elucidate the mechanisms of decreased [18F]-FDG uptake in the AD by PET, [18F]-FDG uptake in the brain of aged model mouse of AD was investigated using a dynamic autoradiography technique "bioradiography". A X-ray phase-contrast imaging (X-PCI) and a histopathological evaluation were also investigated to elucidate the mechanisms underlying the relationships between decreased [18F]-FDG uptake and the pathological changes in the brain of AD mouse. METHODS: In this study, AD model mouse (5XFAD, APP+/PS1+) were used. [18F]-FDG-bioradiography was conducted in fresh slices of brain tissue under the condition of resting (slices immersed in 5 mM K+ solution) and metabolically active (in 50 mM K+ solution). Amyloid ß42 (Aß42) deposition in the brain of AD mouse was confirmed by X-PCI. In addition, the positive cells of phosphated tau protein (P-tau) and deposition of Aß42 were also examined by immunohistochemical staining. RESULTS: No significant differences were observed between the two groups in the resting condition. In the activate condition of the brain, [18F]-FDG uptake was significantly decreased in AD mice compared to WT mice. In X-PCI showed Aß deposition in the AD mouse, but not in the WT. The AD mouse also showed increased P-tau, accumulation of Aß42, increase in neuronal apoptosis, and decrease in the number of neurons than that of the WT mouse. CONCLUSION: Neuronal damage, and induction of neuronal apoptosis, decreased [18F]-FDG uptake, increased Aß accumulation and P-tau induced neurofibrillary degeneration are observed in AD mouse. In clinical diagnosis, reduction of [18F]-FDG uptake by PET is one of the means of diagnosing the onset of AD. Our results suggest that decreased uptake of [18F]-FDG in the brains of AD may be associated with neuronal dysfunction and cell death in the brain.

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.

Ortho-positronium lifetime for soft-tissue classification.

The objective of this work is to showcase the ortho-positronium lifetime as a probe for soft-tissue characterization. We employed positron annihilation lifetime spectroscopy to experimentally measure the three components of the positron annihilation lifetime-para-positronium (p-Ps), positron, and ortho-positronium (o-Ps)-for three types of porcine, non-fixated soft tissues ex vivo: adipose, hepatic, and muscle. Then, we benchmarked our measurements with X-ray phase-contrast imaging, which is the current state-of-the-art for soft-tissue analysis. We found that the o-Ps lifetime in adipose tissues (2.54 ± 0.12 ns) was approximately 20% longer than in hepatic (2.04 ± 0.09 ns) and muscle (2.03 ± 0.12 ns) tissues. In addition, the separation between the measurements for adipose tissue and the other tissues was better from o-Ps lifetime measurement than from X-ray phase-contrast imaging. This experimental study proved that the o-Ps lifetime is a viable non-invasive probe for characterizing and classifying the different soft tissues. Specifically, o-Ps lifetime as a soft-tissue characterization probe had a strong sensitivity to the lipid content that can be potentially implemented in commercial positron emission tomography scanners that feature list-mode data acquisition.