Search

The characterisation of fast phenomena at the microscopic scale is required for the understanding of catastrophic responses of materials to loads and shocks, the processing of materials by optical or mechanical means, the processes involved in many key technologies such as additive manufacturing and microfluidics, and the mixing of fuels in combustion. Such processes are usually stochastic in nature and occur within the opaque interior volumes of materials or samples, with complex dynamics that evolve in all three dimensions at speeds exceeding many meters per second. There is therefore a need for the ability to record three-dimensional X-ray movies of irreversible processes with resolutions of micrometers and frame rates of microseconds. Here we demonstrate a method to achieve this by recording a stereo phase-contrast image pair in a single exposure. The two images are combined computationally to reconstruct a 3D model of the object. The method is extendable to more than two simultaneous views. When combined with megahertz pulse trains of X-ray free-electron lasers (XFELs) it will be possible to create movies able to resolve 3D trajectories with velocities of kilometers per second., Optics Express, 31 (11), ISSN:1094-4087

X-ray grating interferometry CT (GI-CT) is an emerging imaging modality which provides three complementary contrasts that could increase the diagnostic content of clinical breast CT: absorption, phase, and dark-field. Yet, reconstructing the three image channels under clinically compatible conditions is challenging because of severe ill-conditioning of the tomographic reconstruction problem. In this work we propose to solve this problem with a novel reconstruction algorithm that assumes a fixed relation between the absorption and the phase-contrast channel to reconstruct a single image by automatically fusing the absorption and phase channels. The results on both simulations and real data show that, enabled by the proposed algorithm, GI-CT outperforms conventional CT at a clinical dose.

Biophysical models are a promising means for interpreting diffusion weighted magnetic resonance imaging (DW-MRI) data, as they can provide estimates of physiologically relevant parameters of microstructure including cell size, volume fraction, or dispersion. However, their application in cardiac microstructure mapping (CMM) has been limited. This study proposes seven new two-compartment models with combination of restricted cylinder models and a diffusion tensor to represent intra- and extracellular spaces, respectively. Three extended versions of the cylinder model are studied here: cylinder with elliptical cross section (ECS), cylinder with Gamma distributed radii (GDR), and cylinder with Bingham distributed axes (BDA). The proposed models were applied to data in two fixed mouse hearts, acquired with multiple diffusion times, q-shells and diffusion encoding directions. The cylinderGDR-pancake model provided the best performance in terms of root mean squared error (RMSE) reducing it by 25% compared to diffusion tensor imaging (DTI). The cylinderBDA-pancake model represented anatomical findings closest as it also allows for modelling dispersion. High-resolution 3D synchrotron X-ray imaging (SRI) data from the same specimen was utilized to evaluate the biophysical models. A novel tensor-based registration method is proposed to align SRI structure tensors to the MR diffusion tensors. The consistency between SRI and DW-MRI parameters demonstrates the potential of compartment models in assessing physiologically relevant parameters.

X-ray speckle-based imaging (SBI), one of them most recent phase-contrast imaging methods, has received growing interest in the last years. Its simplicity, cost-effectiveness and robustness combined with the high phase sensitivity and compatibility with laboratory X-ray sources make it an attractive method for visualising even minute density differences in samples. Since its first demonstration, SBI has seen rapid development and a range of applications have been identified. Among the various ways to perform SBI, the unified modulated pattern analysis (UMPA) offers a number of advantages. Here, we present an overview of the state of the art of SBI, including some of our work using UMPA in the recent years. We demonstrate the potential of UMPA for applications such as optics characterisation, biomedical and geological imaging and discuss its translation from large-scale synchrotron facilities to the laboratory.

High-contrast, high-resolution imaging of biomedical specimens is indispensable for studying organ function and pathologies. Conventional histology, the gold standard for soft-tissue visualization, is limited by its anisotropic spatial resolution, elaborate sample preparation, and lack of quantitative image information. X-ray absorption or phase tomography have been identified as promising alternatives enabling non-destructive, distortion-free three-dimensional (3D) imaging. However, reaching sufficient contrast and resolution with a simple experimental procedure remains a major challenge. Here, we present a solution based on x-ray phase tomography through speckle-based imaging (SBI). We demonstrate on a mouse kidney that SBI delivers comprehensive 3D maps of hydrated, unstained soft tissue, revealing its microstructure and delivering quantitative tissue-density values at a density resolution of better than 2 m g / c m 3 and spatial resolution of better than 8 µm. We expect that SBI virtual histology will find widespread application in biomedicine and will open up new possibilities for research and histopathology.

Attributingin vivoneurophysiology to the brains’ ultrastructure requires a large field of view containing contextual anatomy. Electron microscopy (EM) is the gold standard technique to identify ultrastructure, yet acquiring volumes containing full mammalian neural circuits is challenging and time consuming using EM. Here, we show that synchrotron X-ray computed tomography (SXRT) provides rapid imaging of EM-prepared tissue volumes of several cubic millimetres. Resolution was sufficient for distinguishing cell bodies as well as for tracing apical dendrites in olfactory bulb and hippocampus, for up to 350 μm. Correlating EM with SXRT allowed us to associate dendritic spines on pyramidal cell apical dendrites in the stratum radiatum to their corresponding soma locations. Superficial pyramidal neurons had larger spine apparatus density compared to deeper ones, implying differential synaptic plasticity for superficial and deeper cells. Finally, we show that X-ray tomography and volume EM can be reliably correlated to priorin vivoimaging. Thus, combining functional measurements with multiscale X-ray microscopy and volume EM establishes a correlative workflow that enables functional and structural investigation of subcellular features in the context of cellular morphologies, tissues and ultimately whole organs.

X-ray grating interferometry (XGI) and speckle-based imaging (SBI) rely on similar principles: The modulations of a reference pattern induced by X-ray refraction, absorption and small-angle scattering in the sample are analysed computationally. The key differences between the methods are the data analysis method and the type of reference pattern, as discussed in more detail in Appendix A. However, it will be shown in Chap. 6 that the two approaches can be unified in a single analysis method that can be applied to both periodic and random reference patterns.

This chapter gives an overview of the working principles, state of the art and recent developments of X-ray speckle-based imaging to establish a background and understanding for the following chapters. As part of this Ph.D. project, the author wrote an extensive literature review on X-ray speckle-based imaging.

As outlined in the previous chapters, X-ray speckle-based imaging has already seen rapid development since its first demonstration less than a decade ago. The rising interest and uptake of the technique calls for its further optimisation and customisation in order to widen its user community and explore new applications.

This Ph.D. project encompasses work on a range of different aspects of X-ray speckle- and single-grating-based phase-contrast imaging.

Following some general information on synchrotron radiation sources at the end of the previous chapter, this chapter outlines the main features and specifications of the beamline I13 at Diamond Light Source and the beamline ID19 at the ESRF, where most of the experiments of this project were carried out.

Most of the implementations of X-ray speckle-based phase tomography reported in the literature to date are proof-of-principle demonstrations and mostly performed on samples of limited scientific interest. A major achievement of this Ph.D. project was the translation of speckle phase tomography to real-world applications, in particular for 3D virtual histology of unstained biomedical soft-tissue specimens. This was enabled by the benefits of the UMPA approach for speckle-based imaging, which was introduced in Chap. 6. UMPA has proven suitable for biomedical imaging applications thanks to its high sensitivity to small density differences, its relaxed requirements on the experimental conditions and its flexible character, amongst others.

Optica 6(9), 1106 - 1109 (2019). doi:10.1364/OPTICA.6.001106, Modern emerging technologies, such as additive manufacturing, bioprinting, and new material production, require novel metrology tools to probe fundamental high-speed dynamics happening in such systems. Here we demonstrate the application of the megahertz (MHz) European X-ray Free-Electron Laser (EuXFEL) to image the fast stochastic processes induced by a laser on water-filled capillaries with micrometer-scale spatial resolution. The EuXFEL provides superior contrast and spatial resolution compared to equivalent state-of-the-art synchrotron experiments. This work opens up new possibilities for the characterization of MHz stochastic processes on the nanosecond to microsecond time scales with object velocities up to a few kilometers per second using XFEL sources., Published by OSA, Washington, DC

X-ray phase-contrast techniques are powerful methods for discerning features with similar densities, which are normally indistinguishable with conventional absorption contrast. While these techniques are well-established tools at large-scale synchrotron facilities, efforts have increasingly focused on implementations at laboratory sources for widespread use. X-ray speckle-based imaging is one of the phase-contrast techniques with high potential for translation to conventional x-ray systems. It yields phase-contrast, transmission, and dark-field images with high sensitivity using a relatively simple and cost-effective setup tolerant to divergent and polychromatic beams. Recently, we have introduced the unified modulated pattern analysis (UMPA) [Phys. Rev. Lett. 118, 203903 (2017)PRLTAO0031-900710.1103/PhysRevLett.118.203903], which further simplifies the translation of x-ray speckle-based imaging to low-brilliance sources. Here, we present the proof-of-principle implementation of UMPA speckle-based imaging at a microfocus liquid-metal-jet x-ray laboratory source.

Supplementary Information

The Diamond Beamline I13L is dedicated to micro- and nano- imaging, with two independently operating branchlines. The imaging branch preforms imaging in real space, with In-line phase contrast imaging and grating interferometry at micrometre resolution and full-field transmission microscopy up to 50nm spatial resolution. Highest spatial resolution is achieved on the coherence branchline, where diffraction imaging methods such as Ptychography and Bragg-CDI are performed. The article provides an update about the experimental capabilities at the beamline with an emphasis on the rapidly evolving ptychography capabilities. The latter has evolved to an user-friendly method with non-expert users able to explore their science without any specific a-priory knowledge.

The DIAMOND beamline I13L is dedicated to multi-scale and multi-modal imaging in real and reciprocal space. The beamline consists of two independently operating experimental stations, located at a distance of more than 200 m from the source. The Imaging Branch performs micro-tomography with in-line phase contrast in the 6-30 keV energy range. In addition, a grating interferometry setup and a full-field microscope for nano-tomography are currently implemented. Other techniques providing high-resolution three-dimensional information, in particular coherent X-ray diffraction, are hosted on the Coherence Branch. All imaging methods are tested to operate with large energy bandwidths and therefore shorter exposure times. To this end, two options are currently used: the so-called 'pink-beam' mode using a reflecting mirror and X-ray filters and monochromatic mode using a multilayer monochromator. The operation mode enables science for in-situ and operando studies across a wide range of scientific areas.

Tumor volume is a parameter used to evaluate the performance of new therapies in lung cancer research. Conventional methods that are used to estimate tumor size in mouse models fail to provide fast and reliable volumetric data for tumors grown non-subcutaneously. Here, we evaluated the use of iodine-staining combined with micro-computed tomography (micro-CT) to estimate the tumor volume of ex vivo tumor-burdened lungs. We obtained fast high spatial resolution three-dimensional information of the lungs, and we demonstrated that iodine-staining highlights tumors and unhealthy tissue. We processed iodine-stained lungs for histopathological analysis with routine hematoxylin and eosin (H&E) staining. We compared the traditional tumor burden estimation performed manually with H&E histological slices with a semi-automated method using micro-CT datasets. In mouse models that develop lung tumors with well precise boundaries, the method that we describe here enables to perform a quick estimation of tumorous tissue volume in micro-CT images. Our method overestimates the tumor burden in tumors surrounded by abnormal tissue, while traditional histopathological analysis underestimates tumor volume. We propose to embed micro-CT imaging to the traditional workflow of tumorous lung analyses in preclinical cancer research as a strategy to obtain a more accurate estimation of the total lung tumor burden.

The I13 imaging and coherence beamline aims for multiscale imaging in the energy range of 6-30keV. The achievable resolution ranges from several microns to tens of nanometers. Several experimental methods are available on two independently operating branchlines. The Diamond-Manchester imaging branchline applies methods in direct space, such as In-line phase contrast, grating interferometry and full-field microscopy. The coherence branch covers far-field techniques such as ptychography and Bragg-CDI. The ptychography is now available as user-friendly experiment and the spatial and temporal resolution has been significantly enhanced. The scientific program at I13 will increasingly explore the suite of experimental techniques available at the beamline for multi-scale imaging.

The Diamond I13 imaging and coherence beamline performs multiscale imaging and tomography with phase sensitive imaging methods. Operating in the energy range of 6-30keV, the achievable resolution ranges from several microns to tens of nanometers [1,2]. The beamline is unique in combining imaging methods in real and reciprocal spaces, which are available on two independently operating branchlines. The scientific program is currently being developed with the distinct capabilities offered by the beamline. Most recently ptycho-tomography has become operational and a multitude of scientific projects is linking the research between both branchlines. The Diamond-Manchester imaging branchline performs mainly in-line phase contrast microtomography with a strong emphasis on dedicated sample environments. Failure of lithium batteries, material cracks, structure of ice cream, bones under load, and storage of CO2 in brine and sandstone are some examples studied under realistic conditions. Most experiments are carried out in the so-called polychromatic 'pink-beam' mode, with micron resolution and millisecond exposure times. Furthermore, a multilayer and a Si (111) monochromator are available for element selective measurements. Two additional projects (full-field microscope and grating interferometry) are currently under development for studies with submicron resolution and phase sensitive imaging. The full-field microscope can perform Zernike phase contrast imaging over a field of view of 50-100µm with a resolution of 50-100nm. The large working distance over several centimeters enable a large variety of scientific applications with dedicated sample environments [3,4]. The different stripes of the multilayer monochromator provide a bandwidth between E/E=10-3 and 10-2 and combined with a fibre-optic plate coupled sCMOS detector, exposure times of few 100ms are achieved. Grating interferometry provides superb image quality, measuring the absolute phase and providing small angle information. We currently plan to apply this method to identify nano-sized structures with micrometer resolution [5,6], as for example in the studies on bones, battery materials, pharmaceutical products or industrial filter materials. Highest spatial resolution is achieved on the coherence branch with ptychographic imaging with 30nm spatial resolution. The photon counting EXCALIBUR detector can continuously record with a speed of up to 250Hz over an array of 1.5x2kPixels. After the last year of development the recording speed for ptycho-tomography scan has significantly improved. A full scan currently takes a few hours and is anticipated to be further accelerated. Ptychography is now performed as standard user experiment, enabling the beamline's ambition for multi-scale imaging. A large number of pilot experiments are being

X-ray phase-contrast and dark-field imaging provides valuable, complementary information about the specimen under study. Among the multimodal X-ray imaging methods, X-ray grating interferometry and speckle-based imaging have drawn particular attention, which, however, in their common implementations incur certain limitations that can restrict their range of applications. Recently, the unified modulated pattern analysis (UMPA) approach was proposed to overcome these limitations and combine grating- and speckle-based imaging in a single approach. Here, we demonstrate the multimodal imaging capabilities of UMPA and highlight its tunable character regarding spatial resolution, signal sensitivity and scan time by using different reconstruction parameters., Journal of Instrumentation, 13, ISSN:1748-0221

International audience; We report on indirect X-ray detector systems for various full-field, ultra high-speed X-ray imaging methodologies, such as X-ray phase-contrast radiography, diffraction topography, grating interferometry and speckle-based imaging performed at the hard X-ray imaging beamline ID19 of the European Synchrotron—ESRF. Our work highlights the versatility of indirect X-ray detectors to multiple goals such as single synchrotron pulse isolation, multiple-frame recording up to millions frames per second, high efficiency, and high spatial resolution. Besides the technical advancements, potential applications are briefly introduced and discussed.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12968-017-0342-x) contains supplementary material, which is available to authorized users.

The Diamond Beamline I13L is dedicated to imaging on the micro- and nano-lengthsale, operating in the energy range between 6 and 30keV. For this purpose two independently operating branchlines and endstations have been built. The imaging branch is fully operational for micro-tomography and in-line phase contrast imaging with micrometre resolution. Grating interferometry is currently implemented, adding the capability of measuring phase and small-angle information. For tomography with increased resolution a full-field microscope providing 50nm spatial resolution with a field of view of 100μm is being tested. The instrument provides a large working distance between optics and sample to adapt a wide range of customised sample environments. On the coherence branch coherent diffraction imaging techniques such as ptychography, coherent X-ray diffraction (CXRD) are currently developed for three dimensional imaging with the highest resolution. The imaging branch is operated in collaboration with Manchester University, called therefore the Diamond-Manchester Branchline. The scientific applications cover a large area including bio-medicine, materials science, chemistry geology and more. The present paper provides an overview about the current status of the beamline and the science addressed.

The Diamond Beamline I13L is designed to imaging on the micron- and nano-lengthsale with X-rays of energies between 6 and 30 keV [1]. Two independently operating branchlines and endstations have been built at distance of more than 200m from the source for this purpose. The imaging branch is dedicated for imaging in real space, providing In-line phase contrast imaging and grating interferometry with micrometre resolution and full-field transmission microscopy with 50nm spatial resolution. On the coherence branch coherent diffraction imaging techniques such as ptychography, coherent X-ray diffraction (CXRD) and Fourier-Transform holography are currently developed. Because of the large lateral coherence length available at I13, the beamline hosts numerous microscopy experiments. The coherence branchline in particular contains a number of unique features. New instrumental designs have been employed such as a robot arm for the detector in diffraction experiments and a photon counting detector for diffraction experiments. The so-called ‘mini-beta’ layout in the straight section of the electron storage ring permits modulating the horizontal source size and therefor the lateral coherence length. We will present the recent progress in coherent imaging at the beamline and the sciences addressed with the instrumental capabilities. Reference: [1] C. Rau, U. Wagner, Z. Pesic, A. De Fanis Physica Status Solidi (a) 208 (11). Issue 11 2522-2525, 2011, 10.1002/pssa.201184272

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.

Histograms of ratios of principal eigenvalues (PDF 238Â kb)

Phase-contrast imaging with x-rays is a developing field for imaging weakly absorbing materials. In this work, two phase-contrast imaging methods, grating- and speckle-based imaging, that measure the derivative of the phase shift, have been implemented with a laboratory source and compared experimentally. It was found that for the same dose conditions, the speckle-tracking differential phase-contrast images have considerably higher contrast-to-noise ratio than the grating-based images, but at the cost of lower resolution. Grating-based imaging performs better in terms of resolution, but would require longer exposure times, mainly due to absorption in the grating interferometer.

Sheetlet elevation and azimuth maps and histograms (PDF 610Â kb)

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. QC 20170614

The phase-stepping (PS) mode of X-ray Grating Talbot interferometer (XGTI) data processing technique can yield high-spatial resolution images, albeit with lower throughput since each projection of a tomogram requires at least five phase-stepping images. To overcome this problem, we can use a setup with only a single phase grating in combination with a high-resolution detector system and a Spatial Harmonic Imaging (SHI) technique. The latter technique makes use of the fact that a Talbot interferogram consists of carrier frequency spectrum of the grating overlapping with the sample spectrum and by Fourier transforming the interferogram we can separate the two. The disadvantage of this is that the spatial resolution is inferior. In this manuscript we will compare these two single grating data processing techniques using a single data set measured with mouse embryo heart and discuss advantages and disadvantages of each technique. These two techniques can be used as complementary; one for high resolution, and the other for high-speed 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.

X-ray imaging in absorption contrast mode is well established for hard tissue visualization. However, performance for lower density materials is limited due to a reduced contrast. Our aim is three-dimensional (3D) characterization of micro-morphology of human brain tissues down to (sub-)cellular resolution within a laboratory environment. Using the laboratory-based microtomography (μCT) system nanotom m (GE Sensing and Inspection Technologies GmbH, Wunstorf, Germany) and synchrotron radiation at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK), we have acquired 3D data with a resolution down to 0.45 μm for visualization of a human cerebellum specimen down to cellular level. We have shown that all selected modalities, namely laboratory-based absorption contrast micro-tomography (LBμCT), synchrotron radiation based in-line single distance phase contrast tomography (SDPR) and synchrotron radiation based single-grating interferometry (GI), can reach cellular resolution for tissue samples with a size in the mm-range. The results are discussed qualitatively in comparison to optical microscopy of haematoxylin and eosin (HE) stained sections. As phase contrast yields to a better data quality for soft tissues and in order to overcome restrictions of limited beamline access for phase contrast measurements, we have equipped the μCT system nanotom m with a double-grating phase contrast set-up. Preliminary experimental results of a knee sample consisting of a bony part and a cartilage demonstrate that phase contrast data exhibits better quality compared to absorption contrast. Currently, the set-up is under adjustment. It is expected that cellular resolution would also be achieved. The questions arise (1) what would be the quality gain of laboratory-based phase contrast in comparison to laboratory-based absorption contrast tomography and (2) could laboratory-based phase contrast data provide comparable results to synchrotron radiation based phase contrast data.

Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.

The Diamond Beamline I13L is dedicated to imaging on the micron- and nano-lengthscale, operating in the energy range between 6 and 30 keV. For this purpose two independent stations have been built. The imaging branch is fully operational for micro-tomography and in-line phase contrast imaging with micrometer resolution. Currently a full-field microscope providing 50nm spatial resolution over a field of view of 100 µm is being tested. On the coherence branch, coherent diffraction imaging techniques such as ptychography and coherent X-ray Bragg diffraction are currently developed. The beamline contains a number of unique features. The machine layout has been modified to the so-called mini-beta scheme, providing significantly increased flux from the two canted undulators. New instrumental designs such as a robot arm for the detector in diffraction experiments have been employed. The imaging branch is operated in collaboration with Manchester University, called therefore the Diamond-Manchester Branchline.

X-ray dark-field and phase-contrast imaging using near-field speckles is a novel technique that overcomes limitations inherent in conventional absorption X-ray imaging, i.e. poor contrast for features with similar density. Speckle-based imaging yields a wealth of information with a simple setup tolerant to polychromatic and divergent beams, and simple data acquisition and analysis procedures. Here, we present a simulation software used to model the image formation with the speckle-based technique, and we compare simulated results on a phantom sample with experimental synchrotron data. Thorough simulation of a speckle-based imaging experiment will help for better understanding and optimising the technique itself.

There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.