Imaging Breast Microcalcifications Using Dark-Field Signal in Propagation-Based Phase-Contrast Tomography.

Breast microcalcifications are an important primary radiological indicator of breast cancer. However, microcalcification classification and diagnosis may be still challenging for radiologists due to limitations of the standard 2D mammography technique, including spatial and contrast resolution. In this study, we propose an approach to improve the detection of microcalcifications in propagation-based phase-contrast X-ray computed tomography of breast tissues. Five fresh mastectomies containing microcalcifications were scanned at different X-ray energies and radiation doses using synchrotron radiation. Both bright-field (i.e. conventional phase-retrieved images) and dark-field images were extracted from the same data sets using different image processing methods. A quantitative analysis was performed in terms of visibility and contrast-to-noise ratio of microcalcifications. The results show that while the signal-to-noise and the contrast-to-noise ratios are lower, the visibility of the microcalcifications is more than two times higher in the dark-field images compared to the bright-field images. Dark-field images have also provided more accurate information about the size and shape of the microcalcifications.

Propagation-based x-ray phase-contrast tomography of mastectomy samples using synchrotron radiation.

PURPOSE: Propagation-based phase-contrast computed tomography (PB-CT) is a method for three-dimensional x-ray imaging that utilizes refraction, as well as absorption, of x rays in the tissues to increase the signal-to-noise ratio (SNR) in the resultant images, in comparison with equivalent conventional absorption-only x-ray tomography (CT). Importantly, the higher SNR is achieved without sacrificing spatial resolution or increasing the radiation dose delivered to the imaged tissues. The present work has been carried out in the context of the current development of a breast CT imaging facility at the Australian Synchrotron. METHODS: Seven unfixed complete mastectomy samples with and without breast cancer lesions have been imaged using absorption-only CT and PB-CT techniques under controlled experimental conditions. The radiation doses delivered to the mastectomy samples during the scans were comparable to those approved for mammographic screening. Physical characteristics of the reconstructed images, such as spatial resolution and SNR, have been measured and compared with the results of the radiological quality assessment of the complete absorption CT and PB-CT image stacks. RESULTS: Despite the presence of some image artefacts, the PB-CT images have outperformed comparable absorption CT images collected at the same radiation dose, in terms of both the measured objective image characteristics and the radiological image scores. The outcomes of these experiments are shown to be consistent with predictions of the theory of PB-CT imaging and previous reported experimental studies of this imaging modality. CONCLUSIONS: The results presented in this paper demonstrate that PB-CT holds a high potential for improving on the quality and diagnostic value of images obtained using existing medical x-ray technologies, such as mammography and digital breast tomosynthesis (DBT). If implemented at suitable synchrotron imaging facilities, PB-CT can be used to complement existing imaging modalities, leading to more accurate breast cancer diagnosis.

Modal approach for partially coherent diffractive imaging with simultaneous sample and coherence recovery.

We demonstrate a modal approach to simultaneous recovery of a sample transmission function and coherence properties of an illuminating X-ray beam that works from a single measurement. The approach based on coherence diffractive imaging, does not depend on a particular model for the coherence function. This single shot imaging method separates the effects of partial coherence in the illuminating beam from the sample, allowing the recovery of high quality sample information.

Characterization of an indirect X-ray imaging detector by simulation and experiment.

We describe a comprehensive model of a commercial indirect X-ray imaging detector that accurately predicts the detector point spread function and its dependence on X-ray energy. The model was validated by measurements using monochromatic synchrotron radiation and extended to polychromatic X-ray sources. Our approach can be used to predict the performance of an imaging detector and can be used to optimize imaging experiments with broad-band X-ray sources.

Application of a complex constraint for biological samples in coherent diffractive imaging.

We demonstrate the application of a complex constraint in the reconstruction of images from phase-diverse Fresnel coherent diffraction data for heterogeneous biological objects. The application of this constraint is shown to improve the quality of the reconstruction of both the phase and the magnitude of the complex object transmission function.

Phase-diverse Fresnel coherent diffractive imaging of malaria parasite-infected red blood cells in the water window.

Phase-diverse Fresnel coherent diffractive imaging has been shown to reveal the structure and composition of biological specimens with high sensitivity at nanoscale resolution. However, the method has yet to be applied using X-ray illumination with energy in the so-called 'water-window' that lies between the carbon and oxygen K edges. In this range, differences in the strength of the X-ray interaction for protein based biological materials and water is increased. Here we demonstrate a proof-of-principle application of FCDI at an X-ray energy within the water-window to a dehydrated cellular sample composed of red blood cells infected with the trophozoite stage of the malaria parasite, Plasmodium falciparum. Comparison of the results to both optical and electron microscopy shows that the correlative imaging methods that include water-window FCDI will find utility in studying cellular architecture.

Nanoscale Fresnel coherent diffraction imaging tomography using ptychography.

We demonstrate Fresnel Coherent Diffractive Imaging (FCDI) tomography in the X-ray regime. The method uses an incident X-ray illumination with known curvature in combination with ptychography to overcome existing problems in diffraction imaging. The resulting tomographic reconstruction represents a 3D map of the specimen's complex refractive index at nano-scale resolution. We use this technique to image a lithographically fabricated glass capillary, in which features down to 70nm are clearly resolved.

Simultaneous X-ray fluorescence and ptychographic microscopy of Cyclotella meneghiniana.

Scanning X-ray fluorescence microscopy (XFM) is a particularly useful method for studying the spatial distribution of trace metals in biological samples. Here we demonstrate the utility of combining coherent diffractive imaging (CDI) with XFM for imaging biological samples to simultaneously produce high-resolution and high-contrast transmission images and quantitative elemental maps. The reconstructed transmission function yields morphological details which contextualise the elemental maps. We report enhancement of the spatial resolution in both the transmission and fluorescence images beyond that of the X-ray optics. The freshwater diatom Cyclotella meneghiniana was imaged to demonstrate the benefits of combining these techniques that have complementary contrast mechanisms.

An in-vacuum x-ray diffraction microscope for use in the 0.7-2.9 keV range.

A dedicated in-vacuum coherent x-ray diffraction microscope was installed at the 2-ID-B beamline of the Advanced Photon Source for use with 0.7-2.9 keV x-rays. The instrument can accommodate three common implementations of diffractive imaging; plane wave illumination; defocused-probe (Fresnel diffractive imaging) and scanning (ptychography) using either a pinhole, focused or defocused probe. The microscope design includes active feedback to limit motion of the optics with respect to the sample. Upper bounds on the relative optics-to-sample displacement have been measured to be 5.8 nm(v) and 4.4 nm(h) rms/h using capacitance micrometry and 27 nm/h using x-ray point projection imaging. The stability of the measurement platform and in-vacuum operation allows for long exposure times, high signal-to-noise and large dynamic range two-dimensional intensity measurements to be acquired. Finally, we illustrate the microscope's stability with a recent experimental result.

Coherence properties of individual femtosecond pulses of an x-ray free-electron laser.

Measurements of the spatial and temporal coherence of single, femtosecond x-ray pulses generated by the first hard x-ray free-electron laser, the Linac Coherent Light Source, are presented. Single-shot measurements were performed at 780 eV x-ray photon energy using apertures containing double pinholes in "diffract-and-destroy" mode. We determined a coherence length of 17 µm in the vertical direction, which is approximately the size of the focused Linac Coherent Light Source beam in the same direction. The analysis of the diffraction patterns produced by the pinholes with the largest separation yields an estimate of the temporal coherence time of 0.55 fs. We find that the total degree of transverse coherence is 56% and that the x-ray pulses are adequately described by two transverse coherent modes in each direction. This leads us to the conclusion that 78% of the total power is contained in the dominant mode.

Imaging the 3D structure of secondary osteons in human cortical bone using phase-retrieval tomography.

By applying a phase-retrieval step before carrying out standard filtered back-projection reconstructions in tomographic imaging, we were able to resolve structures with small differences in density within a densely absorbing sample. This phase-retrieval tomography is particularly suited for the three-dimensional segmentation of secondary osteons (roughly cylindrical structures) which are superimposed upon an existing cortical bone structure through the process of turnover known as remodelling. The resulting images make possible the analysis of the secondary osteon structure and the relationship between an osteon and the surrounding tissue. Our observations have revealed many different and complex 3D structures of osteons that could not be studied using previous methods. This work was carried out using a laboratory-based x-ray source, which makes obtaining these sorts of images readily accessible.

Visualization of 3D osteon morphology by synchrotron radiation micro-CT.

Cortical bone histology has been the subject of scientific inquiry since the advent of the earliest microscopes. Histology - literally the study of tissue - is a field nearly synonymous with 2D thin sections. That said, progressive developments in high-resolution X-ray imaging are enabling 3D visualization to reach ever smaller structures. Micro-computed tomography (micro-CT), employing conventional X-ray sources, has become the gold standard for 3D analysis of trabecular bone and is capable of detecting the structure of vascular (osteonal) porosity in cortical bone. To date, however, direct 3D visualization of secondary osteons has eluded micro-CT based upon absorption-derived contrast. Synchrotron radiation micro-CT, through greater image quality, resolution and alternative contrast mechanisms (e.g. phase contrast), holds great potential for non-destructive 3D visualization of secondary osteons. Our objective was to demonstrate this potential and to discuss areas of bone research that can be advanced through the application of this approach. We imaged human mid-femoral cortical bone specimens derived from a 20-year-old male (Melbourne Femur Collection) at the Advanced Photon Source synchrotron (Chicago, IL, USA) using the 2BM beam line. A 60-mm distance between the target and the detector was employed to enhance visualization of internal structures through propagation phase contrast. Scan times were 1 h and images were acquired with 1.4-µm nominal isotropic resolution. Computer-aided manual segmentation and volumetric 3D rendering were employed to visualize secondary osteons and porous structures, respectively. Osteonal borders were evident via two contrast mechanisms. First, relatively new (hypomineralized) osteons were evident due to differences in X-ray attenuation relative to the surrounding bone. Second, osteon boundaries (cement lines) were delineated by phase contrast. Phase contrast also enabled the detection of soft tissue remnants within the vascular pores. The ability to discern osteon boundaries in conjunction with vascular and cellular porosity revealed a number of secondary osteon morphologies and provided a unique 3D perspective of the superimposition of secondary osteons on existing structures. Improvements in resolution and optimization of the propagation phase contrast promise to provide further improvements in structural detail in the future.

Optimisation of phase imaging geometry.

Certain phase retrieval methods use knowledge about the free space propagation of a wave to phase a paraxial beam passing through one or more measurement planes. This approach has been widely applied and has been shown to quantitatively retrieve the refractive index profile of a sample. The quality of the phase retrieval will depend on a range of factors including sample feature size, propagation distance, measurement plane separation, wavelength and noise. Here we describe an optimisation study for two-plane phase retrieval using a laboratory-based X-ray source that considers all of these factors. We discuss our results in the context of a three-dimensional reconstruction of a sample refractive index profile.

Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT.

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 µm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4×10(4)per mm(3) close to the Haversian canals to about 9×10(4)per mm(3) at 80% of osteon radius. The nearest-neighbour distances varied from 10 µm to 40 µm with a peak at 23 µm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 µm with a range from 3 µm to 20 µm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 µm(3) and a second at 330 µm(3).

Fresnel coherent diffraction tomography.

Tomographic coherent imaging requires the reconstruction of a series of two-dimensional projections of the object. We show that using the solution for the image of one projection as the starting point for the reconstruction of the next projection offers a reliable and rapid approach to the image reconstruction. The method is demonstrated on simulated and experimental data. This technique also simplifies reconstructions using data with curved incident wavefronts.

Use of a complex constraint in coherent diffractive imaging.

We demonstrate use of a complex constraint based on the interaction of x-rays with matter for reconstructing images from coherent X-ray diffraction. We show the complementary information provided by the phase and magnitude of the reconstructed wavefield greatly improves the quality of the resulting estimate of the transmission function of an object without the need for a priori information about the object composition.

A coherence approach to phase-contrast microscopy II: experiment.

We report an experimental investigation of the optical transfer functions for an X-ray microscope operated in defocus phase-contrast mode. The results are compared with a theoretical model of partially coherent image formation and are found to be in excellent agreement.

Diffractive imaging using partially coherent x rays.

The measured spatial coherence characteristics of the illumination used in a diffractive imaging experiment are incorporated in an algorithm that reconstructs the complex transmission function of an object from experimental x-ray diffraction data using 1.4 keV x rays. Conventional coherent diffractive imaging, which assumes full spatial coherence, is a limiting case of our approach. Even in cases in which the deviation from full spatial coherence is small, we demonstrate a significant improvement in the quality of wave field reconstructions. Our formulation is applicable to x-ray and electron diffraction imaging techniques provided that the spatial coherence properties of the illumination are known or can be measured.

Phase imaging using a polychromatic x-ray laboratory source.

We describe a quantitative phase imaging process using an x-ray laboratory-based source with an extremely broad bandwidth spectrum. The thickness of a homogeneous object can be retrieved by using separately spectrally weighted values for the attenuation coefficient and the decrement of the real part of the refractive index. This method is valid for a wide range of object types, including objects with an absorption edge in the used energy range. The accessibility of conventional x-ray laboratory sources makes this method very useful for quantitative phase retrieval of homogeneous objects. We demonstrate the application of this method for quantitative phase retrieval imaging in tomographic measurements.

Quantitative phase measurement in coherent diffraction imaging.

We demonstrate high spatial resolution phase retrieval of a non-periodic gold nano-structure using the method of Fresnel coherent diffractive imaging. The result is quantitative to better than 10% and does not rely on any a priori knowledge of the sample.