Multi-resolution X-ray phase-contrast and dark-field tomography of human cerebellum with near-field speckles.

In this study, we use synchrotron-based multi-modal X-ray tomography to examine human cerebellar tissue in three dimensions at two levels of spatial resolution (2.3 µm and 11.9 µm). We show that speckle-based imaging (SBI) produces results that are comparable to propagation-based imaging (PBI), a well-established phase-sensitive imaging method. The different SBI signals provide complementary information, which improves tissue differentiation. In particular, the dark-field signal aids in distinguishing tissues with similar average electron density but different microstructural variations. The setup's high resolution and the imaging technique's excellent phase sensitivity enabled the identification of different cellular layers and additionally, different cell types within these layers. We also correlated this high-resolution phase-contrast information with measured dark-field signal levels. These findings demonstrate the viability of SBI and the potential benefit of the dark-field modality for virtual histology of brain tissue.

At-wavelength optics characterisation via X-ray speckle- and grating-based unified modulated pattern analysis.

The current advances in new generation X-ray sources are calling for the development and improvement of high-performance optics. Techniques for high-sensitivity phase sensing and wavefront characterisation, preferably performed at-wavelength, are increasingly required for quality control, optimisation and development of such devices. We here show that the recently proposed unified modulated pattern analysis (UMPA) can be used for these purposes. We characterised two polymer X-ray refractive lenses and quantified the effect of beam damage and shape errors on their refractive properties. Measurements were performed with two different setups for UMPA and validated with conventional X-ray grating interferometry. Due to its adaptability to different setups, the ease of implementation and cost-effectiveness, we expect UMPA to find applications for high-throughput quantitative optics characterisation and wavefront sensing.

X-ray Phase-Contrast Imaging and Metrology through Unified Modulated Pattern Analysis.

We present a method for x-ray phase-contrast imaging and metrology applications based on the sample-induced modulation and subsequent computational demodulation of a random or periodic reference interference pattern. The proposed unified modulated pattern analysis (UMPA) technique is a versatile approach and allows tuning of signal sensitivity, spatial resolution, and scan time. We characterize the method and demonstrate its potential for high-sensitivity, quantitative phase imaging, and metrology to overcome the limitations of existing methods.

X-ray phase microtomography with a single grating for high-throughput investigations of biological tissue.

The high-throughput 3D visualisation of biological specimens is essential for studying diseases and developmental disorders. It requires imaging methods that deliver high-contrast, high-resolution volumetric information at short sample preparation and acquisition times. Here we show that X-ray phase-contrast tomography using a single grating can provide a powerful alternative to commonly employed techniques, such as high-resolution episcopic microscopy (HREM). We present the phase tomography of a mouse embryo in paraffin obtained with an X-ray single-grating interferometer at I13-2 Beamline at Diamond Light Source and discuss the results in comparison with HREM measurements. The excellent contrast and quantitative density information achieved non-destructively and without staining using a simple, robust setup make X-ray single-grating interferometry an optimum candidate for high-throughput imaging of biological specimens as an alternative for existing methods like HREM.

Validation of diffusion tensor MRI measurements of cardiac microstructure with structure tensor synchrotron radiation imaging.

BACKGROUND: Diffusion tensor imaging (DTI) is widely used to assess tissue microstructure non-invasively. Cardiac DTI enables inference of cell and sheetlet orientations, which are altered under pathological conditions. However, DTI is affected by many factors, therefore robust validation is critical. Existing histological validation is intrinsically flawed, since it requires further tissue processing leading to sample distortion, is routinely limited in field-of-view and requires reconstruction of three-dimensional volumes from two-dimensional images. In contrast, synchrotron radiation imaging (SRI) data enables imaging of the heart in 3D without further preparation following DTI. The objective of the study was to validate DTI measurements based on structure tensor analysis of SRI data. METHODS: One isolated, fixed rat heart was imaged ex vivo with DTI and X-ray phase contrast SRI, and reconstructed at 100 µm and 3.6 µm isotropic resolution respectively. Structure tensors were determined from the SRI data and registered to the DTI data. RESULTS: Excellent agreement in helix angles (HA) and transverse angles (TA) was observed between the DTI and structure tensor synchrotron radiation imaging (STSRI) data, where HADTI-STSRI = -1.4° ± 23.2° and TADTI-STSRI = -1.4° ± 35.0° (mean ± 1.96 standard deviation across all voxels in the left ventricle). STSRI confirmed that the primary eigenvector of the diffusion tensor corresponds with the cardiomyocyte long-axis across the whole myocardium. CONCLUSIONS: We have used STSRI as a novel and high-resolution gold standard for the validation of DTI, allowing like-with-like comparison of three-dimensional tissue structures in the same intact heart free of distortion. This represents a critical step forward in independently verifying the structural basis and informing the interpretation of cardiac DTI data, thereby supporting the further development and adoption of DTI in structure-based electro-mechanical modelling and routine clinical applications.

Mass Density Measurement of Mineralized Tissue with Grating-Based X-Ray Phase Tomography.

Establishing the mineral content distribution in highly mineralized tissues, such as bones and teeth, is fundamental in understanding a variety of structural questions ranging from studies of the mechanical properties to improved pathological investigations. However, non-destructive, volumetric and quantitative density measurements of mineralized samples, some of which may extend several mm in size, remain challenging. Here, we demonstrate the potential of grating-based x-ray phase tomography to gain insight into the three-dimensional mass density distribution of tooth tissues in a non-destructive way and with a sensitivity of 85 mg/cm3. Density gradients of 13 - 19% over 1 - 2 mm within typical samples are detected, and local variations in density of 0.4 g/cm3 on a length scale of 0.1 mm are revealed. This method proves to be an excellent quantitative tool for investigations of subtle differences in mineral content of mineralized tissues that can change following treatment or during ageing and healing.

Noise analysis of speckle-based x-ray phase-contrast imaging.

Speckle-based x-ray phase-contrast imaging has drawn increasing interest in recent years as a simple, multimodal, cost-efficient, and laboratory-source adaptable method. We investigate its noise properties to help further optimization on the method and further comparison with other phase-contrast methods. An analytical model for assessing noise in a differential phase signal is adapted from studies on the digital image correlation technique in experimental mechanics and is supported by simulations and experiments. The model indicates that the noise of the differential phase signal from speckle-based imaging has a behavior similar to that of the grating-based method.

Hard X-ray phase-contrast tomography of non-homogeneous specimens: grating interferometry versus propagation-based imaging.

X-ray phase-contrast imaging is an effective approach to drastically increase the contrast and sensitivity of microtomographic techniques. Numerous approaches to depict the real part of the complex-valued refractive index of a specimen are nowadays available. A comparative study using experimental data from grating-based interferometry and propagation-based phase contrast combined with single-distance phase retrieval applied to a non-homogeneous sample is presented (acquired at beamline ID19-ESRF). It is shown that grating-based interferometry can handle density gradients in a superior manner. The study underlines the complementarity of the two techniques for practical applications.

Dentinal tubules revealed with X-ray tensor tomography.

Dentin is a mineralized material making up most of the tooth bulk. A system of microtubules, so called dentinal tubules, transverses it radially from the pulp chamber to the outside. This highly oriented structure leads to anisotropic mechanical properties directly connected to the tubules orientation and density: the ultimate tensile strength as well as the fracture toughness and the shear strength are largest perpendicular to dentinal tubules. Consequently, the fatigue strength depends on the direction of dentinal tubules, too. However, none of the existing techniques used to investigate teeth provide access to orientation and density of dentinal tubules for an entire specimen in a non-destructive way. In this paper, we measure a third molar human tooth both with conventional micro-CT and X-ray tensor tomography (XTT). While the achievable resolution in micro-CT is too low to directly resolve the dentinal tubules, we provide strong evidence that the direction and density of dentinal tubules can be indirectly measured by XTT, which exploits small-angle X-ray scattering to retrieve a 3D map of scattering tensors. We show that the mean directions of scattering structures correlate to the orientation of dentinal tubules and that the mean effective scattering strength provides an estimation of the relative density of dentinal tubules. Thus, this method could be applied to investigate the connection between tubule orientation and fatigue or tensile properties of teeth for a full sample without cutting one, non-representative peace of tooth out of the full sample.

Optimization of propagation-based phase-contrast imaging at a laboratory setup.

Single distance X-ray propagation-based phase-contrast imaging is considered as a simple method compared to those requiring additional precise instruments and sophisticated algorithms to retrieve phase images. It requires, however, a modicum of conditions within the setup which include partial coherence and small pixel size at the sample position. While these conditions are usually satisfied at synchrotron light sources, they are not always satisfied within laboratory setups. In fact, these setups are limited by the size of the polychromatic source that directly influences the partial coherence of the beam, the propagation distance and the photon flux. A prior knowledge of the sample refractive index, namely the ratio of delta (δ) and beta (ß) values, are also essential for the phase retrieval but this method is powerful in the presence of noise compared to absorption-based imaging. An investigation of the feasibility and the efficient applicability of this method in a commercially available X-ray microscope is conducted in this work.

Lens-term- and edge-effect in X-ray grating interferometry.

X-ray grating interferometry requires gratings with periods in the micrometer range and allows the acquisition of the dark-field contrast. The analyzer grating is designed to match the period of the interference pattern in order to translate it into a measurable intensity modulation. In this study, we explore the influence of a sample-induced mismatch between the interference pattern and the analyzer grating on the dark-field contrast. We propose a formula for the calculation of the signal due to a period mismatch and present estimations varying periods and detector pixel size. Furthermore, numerical simulations of the X-ray wave-front demonstrate that the wave-front curvature, described by the lens-term, e.g. behind a parabolic lens or edges of a sample can change the period of the interference pattern. Our results give a concrete explanation for the formation of a dark-field contrast from object edges and thus allow a better understanding of the dark-field signal obtained with a grating interferometer.

X-ray microtomography using correlation of near-field speckles for material characterization.

Nondestructive microscale investigation of objects is an invaluable tool in life and materials sciences. Currently, such investigation is mainly performed with X-ray laboratory systems, which are based on absorption-contrast imaging and cannot access the information carried by the phase of the X-ray waves. The phase signal is, nevertheless, of great value in X-ray imaging as it is complementary to the absorption information and in general more sensitive to visualize features with small density differences. Synchrotron facilities, which deliver a beam of high brilliance and high coherence, provide the ideal condition to develop such advanced phase-sensitive methods, but their access is limited. Here we show how a small modification of a laboratory setup yields simultaneously quantitative and 3D absorption and phase images of the object. This single-shot method is based on correlation of X-ray near-field speckles and represents a significant broadening of the capabilities of laboratory-based X-ray tomography.

Characterization of near-field ptychography.

Near-field X-ray ptychography has recently been proposed and shown to be able to retrieve a sample's complex-valued transmission function from multiple near-field diffraction images each with a lateral shift of the sample and with a structured (by a diffuser) illumination [Stockmar et al. Sci Rep. 3 (2013)]. In this paper, we undertake the first investigation - via numerical simulation - of the influence of the sampling and step size of the lateral shifts, the diffuser structure size, and the propagation distance on the reconstruction of the sample's transmission function. We find that for a gold Siemens star of thickness 750 nm with typical experimental parameters, for a successful reconstruction - given a theoretical minimum of four required measurements per imaged pixel - at least six diffraction images are required.

Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography.

Low inherent contrast in soft tissues has been limiting the use of X-ray absorption micro-computed tomography (micro-CT) to access high-resolution structural information of animal organs. The staining agents used in micro-CT to improve the contrast fail in providing high-quality images of whole organs of animals due to diffusion problems of the staining agent into the sample. We demonstrate a staining protocol that incorporates a biochemical conditioning step prior to exposure to the staining agent that succeeds in overcoming the diffusion problems, thus quickly providing high-quality micro-CT images of whole organs of mammals. Besides of yielding non-distorted three-dimensional information at the same spatial resolution accessible in histological sections, micro-CT images of whole organs stained by our method enable easy screening of slices along any direction of the volume thus demonstrating new possibilities of structural analysis in biomedical science.

X-ray nanotomography using near-field ptychography.

Propagation-based imaging or inline holography in combination with computed tomography (holotomography) is a versatile tool to access a sample's three-dimensional (3D) micro or nano structure. However, the phase retrieval step needed prior to tomographic reconstruction can be challenging especially for strongly absorbing and refracting samples. Near-field ptychography is a recently developed phase imaging method that has been proven to overcome this hurdle in projection data. In this work we extend near-field ptychography to three dimensions and we show that, in combination with tomography, it can access the nano structure of a solid oxide fuel cell (SOFC). The quality of the resulting tomographic data and the structural properties of the anode extracted from this volume were compared to previous results obtained with holotomography. This work highlights the potential of 3D near-field ptychography for reliable and detailed investigations of samples at the nanometer scale, with important applications in materials and life sciences among others.

Speckle-based x-ray phase-contrast imaging with a laboratory source and the scanning technique.

The speckle-based scanning method for x-ray phase-contrast imaging is implemented with a liquid-metal-jet source. Using the two-dimensional scanning technique, the phase shift introduced by the object is retrieved in both transverse orientations, and the limitations on spatial resolution inherent to the speckle-tracking technique are avoided. This method opens up possibilities of new high-resolution multimodal applications for lab-based phase-contrast x-ray imaging.

X-ray phase-contrast tomography of renal ischemia-reperfusion damage.

PURPOSE: The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation. MATERIAL AND METHODS: The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 µm. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed. RESULTS: GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders. CONCLUSION: In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney.

Complementary X-ray tomography techniques for histology-validated 3D imaging of soft and hard tissues using plaque-containing blood vessels as examples.

A key problem in X-ray computed tomography is choosing photon energies for postmortem specimens containing both soft and hard tissues. Increasing X-ray energy reduces image artifacts from highly absorbing hard tissues including plaque, but it simultaneously decreases contrast in soft tissues including the endothelium. Therefore, identifying the lumen within plaque-containing vessels is challenging. Destructive histology, the gold standard for tissue evaluation, reaches submicron resolution in two dimensions, whereas slice thickness limits spatial resolution in the third. We present a protocol to systematically analyze heterogeneous tissues containing weakly and highly absorbing components in the original wet state, postmortem. Taking the example of atherosclerotic human coronary arteries, the successively acquired 3D data of benchtop and synchrotron radiation-based tomography are validated by histology. The entire protocol requires ∼20 working days, enables differentiation between plaque, muscle and fat tissues without using contrast agents and permits blood flow simulations in vessels with plaque-induced constrictions.

X-ray phase-contrast tomosynthesis for improved breast tissue discrimination.

PURPOSE: Attenuation-based tomosynthesis has proven to successfully resolve the glandular tissue overlap present in mammography. However, the ability of tomosynthesis to differentiate tumorous and glandular tissue remains limited, due to the small differences in X-ray attenuation in breast tissue. One possibility to overcome this limitation and to further increase the diagnostic value of tomosynthesis exams, is the application of recently developed grating-based phase-contrast methods, which provide complementary information on the phase shift and the local scattering power of the sample. In this study, we report on first phase-contrast breast tomosynthesis results of a mastectomy sample slice with an invasive ductal carcinoma. MATERIAL AND METHODS: A slice of a mastectomy sample with histologically proven invasive ductal cancer was imaged at the synchrotron radiation source ESRF (Grenoble, France). We used a two-grating interferometer setup at the ninth fractional Talbot distance and with an X-ray energy of 23 keV. In grating interferometry absorption, differential phase, and scattering images are recorded simultaneously. The tomosynthesis scan comprises 61 projections. Multimodal tomosynthesis results were reconstructed using a standard filtered back-projection approach. Our findings are supported by a comparison of tomographic views to histopathology. RESULTS: Phase-contrast tomosynthesis combines the advantage of improved soft-tissue discrimination in phase-contrast imaging with the ability of tomosynthesis to provide a third dimension so that improved feature visibility is not hampered by superposition artifacts. Our results indicate superior diagnostic value due to the depth resolution supplied in tomosynthesis imaging; a region of necrotic tissue that is obscured in a projection image can clearly be depicted in one single tomosynthesis slice. Compared to absorption tomosynthesis alone, soft tissue contrast is significantly enhanced in phase-contrast tomosynthesis views, where fibrous structures are clearly visible. CONCLUSION: In this article we present the first proof-of-principle grating-based phase-contrast tomosynthesis of a mastectomy sample section. A comparison of conventional attenuation with phase-contrast and dark-field tomosynthesis indicates that complementary information from three signals yields an increase in diagnostic value, which is verified in a comparison of our results to histological sections of the sample. As grating-based phase-contrast mammography efficiently works with conventional lab sources, our benchmark results indicate the potential benefit of translating phase-contrast tomosynthesis into a clinical setting.

Translation of atherosclerotic plaque phase-contrast CT imaging from synchrotron radiation to a conventional lab-based X-ray source.

OBJECTIVES: Phase-contrast imaging is a novel X-ray based technique that provides enhanced soft tissue contrast. The aim of this study was to evaluate the feasibility of visualizing human carotid arteries by grating-based phase-contrast tomography (PC-CT) at two different experimental set-ups: (i) applying synchrotron radiation and (ii) using a conventional X-ray tube. MATERIALS AND METHODS: Five ex-vivo carotid artery specimens were examined with PC-CT either at the European Synchrotron Radiation Facility using a monochromatic X-ray beam (2 specimens; 23 keV; pixel size 5.4 µm), or at a laboratory set-up on a conventional X-ray tube (3 specimens; 35-40 kVp; 70 mA; pixel size 100 µm). Tomographic images were reconstructed and compared to histopathology. Two independent readers determined vessel dimensions and one reader determined signal-to-noise ratios (SNR) between PC-CT and absorption images. RESULTS: In total, 51 sections were included in the analysis. Images from both set-ups provided sufficient contrast to differentiate individual vessel layers. All PCI-based measurements strongly predicted but significantly overestimated lumen, intima and vessel wall area for both the synchrotron and the laboratory-based measurements as compared with histology (all p<0.001 with slope >0.53 per mm(2), 95%-CI: 0.35 to 0.70). Although synchrotron-based images were characterized by higher SNRs than laboratory-based images; both PC-CT set-ups had superior SNRs compared to corresponding conventional absorption-based images (p<0.001). Inter-reader reproducibility was excellent (ICCs >0.98 and >0.84 for synchrotron and for laboratory-based measurements; respectively). CONCLUSION: Experimental PC-CT of carotid specimens is feasible with both synchrotron and conventional X-ray sources, producing high-resolution images suitable for vessel characterization and atherosclerosis research.