Feasibility of Dark-Field Radiography to Enhance Detection of Nondisplaced Fractures.

Background Many clinically relevant fractures are occult on conventional radiographs and therefore challenging to diagnose reliably. X-ray dark-field radiography is a developing method that uses x-ray scattering as an additional signal source. Purpose To investigate whether x-ray dark-field radiography enhances the depiction of radiographically occult fractures in an experimental model compared with attenuation-based radiography alone and whether the directional dependence of dark-field signal impacts observer ratings. Materials and Methods Four porcine loin ribs had nondisplaced fractures experimentally introduced. Microstructural changes were visually verified using high-spatial-resolution three-dimensional micro-CT. X-ray dark-field radiographs were obtained before and after fracture, with the before-fracture scans serving as control images. The presence of a fracture was scored by three observers using a six-point scale (6, surely; 5, very likely; 4, likely; 3, unlikely; 2, very unlikely; and 1, certainly not). Differences between scores based on attenuation radiographs alone (n = 96) and based on combined attenuation and dark-field radiographs (n = 96) were evaluated by using the DeLong method to compare areas under the receiver operating characteristic curve. The impact of the dark-field signal directional sensitivity on observer ratings was evaluated using the Wilcoxon test. The dark-field data were split into four groups (24 images per group) according to their sensitivity orientation and tested against each other. Musculoskeletal dark-field radiography was further demonstrated on human finger and foot specimens. Results The addition of dark-field radiographs was found to increase the area under the receiver operating characteristic curve to 1 compared with an area under the receiver operating characteristic curve of 0.87 (95% CI: 0.80, 0.94) using attenuation-based radiographs alone (P < .001). There were similar observer ratings for the four different dark-field sensitivity orientations (P = .16-.65 between the groups). Conclusion These results suggested that the inclusion of dark-field radiography has the potential to help enhance the detection of nondisplaced fractures compared with attenuation-based radiography alone. © RSNA, 2024 See also the editorial by Rubin in this issue.

Simultaneous two-color X-ray absorption spectroscopy using Laue crystals at an inverse-compton scattering X-ray facility.

X-ray absorption spectroscopy (XAS) is an element-selective technique that provides electronic and structural information of materials and reveals the essential mechanisms of the reactions involved. However, the technique is typically conducted at synchrotrons and usually only probes one element at a time. In this paper, a simultaneous two-color XAS setup at a laboratory-scale synchrotron facility is proposed based on inverse Compton scattering (ICS) at the Munich Compact Light Source (MuCLS), which is based on inverse Compton scattering (ICS). The setup utilizes two silicon crystals in a Laue geometry. A proof-of-principle experiment is presented where both silver (Ag) and palladium (Pd) K-edge X-ray absorption near-edge structure spectra were simultaneously measured. The simplicity of the setup facilitates its migration to other ICS facilities or maybe to other X-ray sources (e.g. a bending-magnet beamline). Such a setup has the potential to study reaction mechanisms and synergistic effects of chemical systems containing multiple elements of interest, such as a bimetallic catalyst system.

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.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of Euperipatoides rowelli, a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

A proof of principle experiment for microbeam radiation therapy at the Munich compact light source.

Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.

Multimodal Precision Imaging of Pulmonary Nanoparticle Delivery in Mice: Dynamics of Application, Spatial Distribution, and Dosimetry.

Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.

Device for source position stabilization and beam parameter monitoring at inverse Compton X-ray sources.

Compact X-ray sources based on inverse Compton scattering provide brilliant and partially coherent X-rays in a laboratory environment. The cross section for inverse Compton scattering is very small, requiring high-power laser systems as well as small laser and electron beam sizes at the interaction point to generate sufficient flux. Therefore, these systems are very sensitive to distortions which change the overlap between the two beams. In order to monitor X-ray source position, size and flux in parallel to experiments, the beam-position monitor proposed here comprises a small knife edge whose image is acquired with an X-ray camera specifically designed to intercept only a very small fraction of the X-ray beam. Based on the source position drift recorded with the monitor, a closed-loop feedback stabilizes the X-ray source position by adjusting the laser beam trajectory. A decrease of long-term source position drifts by more than one order of magnitude is demonstrated with this device. Consequently, such a closed-loop feedback system which enables stabilization of source position drifts and flux of inverse Compton sources in parallel to experiments has a significant impact on the performance of these sources.

The Munich Compact Light Source: initial performance measures.

While large-scale synchrotron sources provide a highly brilliant monochromatic X-ray beam, these X-ray sources are expensive in terms of installation and maintenance, and require large amounts of space due to the size of storage rings for GeV electrons. On the other hand, laboratory X-ray tube sources can easily be implemented in laboratories or hospitals with comparatively little cost, but their performance features a lower brilliance and a polychromatic spectrum creates problems with beam hardening artifacts for imaging experiments. Over the last decade, compact synchrotron sources based on inverse Compton scattering have evolved as one of the most promising types of laboratory-scale X-ray sources: they provide a performance and brilliance that lie in between those of large-scale synchrotron sources and X-ray tube sources, with significantly reduced financial and spatial requirements. These sources produce X-rays through the collision of relativistic electrons with infrared laser photons. In this study, an analysis of the performance, such as X-ray flux, source size and spectra, of the first commercially sold compact light source, the Munich Compact Light Source, is presented.

Ptychographic X-ray computed tomography at the nanoscale.

X-ray tomography is an invaluable tool in biomedical imaging. It can deliver the three-dimensional internal structure of entire organisms as well as that of single cells, and even gives access to quantitative information, crucially important both for medical applications and for basic research. Most frequently such information is based on X-ray attenuation. Phase contrast is sometimes used for improved visibility but remains significantly harder to quantify. Here we describe an X-ray computed tomography technique that generates quantitative high-contrast three-dimensional electron density maps from phase contrast information without reverting to assumptions of a weak phase object or negligible absorption. This method uses a ptychographic coherent imaging approach to record tomographic data sets, exploiting both the high penetration power of hard X-rays and the high sensitivity of lensless imaging. As an example, we present images of a bone sample in which structures on the 100 nm length scale such as the osteocyte lacunae and the interconnective canalicular network are clearly resolved. The recovered electron density map provides a contrast high enough to estimate nanoscale bone density variations of less than one per cent. We expect this high-resolution tomography technique to provide invaluable information for both the life and materials sciences.

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.

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.

Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction.

The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal Fe─O (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of ß-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerol-water interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper.

High-energy X-ray diffraction experiment employing a compact synchrotron X-ray source based on inverse Compton scattering.

X-ray diffraction (XRD) is an important material analysis technique with a widespread use of laboratory systems. These systems typically operate at low X-ray energies (from 5 keV to 22 keV) since they rely on the small bandwidth of K-lines like copper. The narrow bandwidth is essential for precise measurements of the crystal structure in these systems. Inverse Compton X-ray source (ICS) could pave the way to XRD at high X-ray energies in a laboratory setting since these sources provide brilliant energy-tunable and partially coherent X-rays. This study demonstrates high-energy XRD at an ICS with strongly absorbing mineralogical samples embedded in soft tissue. A quantitative comparison of the measured XRD patterns with calculations of their expected shapes validates the performance of ICSs for XRD. This analysis was performed for two types of kidney stones of different materials. Since these stones are not isolated in a human body, the influence of the surrounding soft tissue on the XRD pattern is investigated and a correction for this soft tissue contribution is introduced.

Quantitative biological imaging by ptychographic x-ray diffraction microscopy.

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of approximately 1.3x10(5) Gy, and were shown to scale with photon fluence.

X-ray Stain Localization with Near-Field Ptychographic Computed Tomography.

Although X-ray contrast agents offer specific characteristics in terms of targeting and attenuation, their accumulation in the tissue on a cellular level is usually not known and difficult to access, as it requires high resolution and sensitivity. Here, quantitative near-field ptychographic X-ray computed tomography is demonstrated to assess the location of X-ray stains at a resolution sufficient to identify intracellular structures by means of a basis material decomposition. On the example of two different X-ray stains, the nonspecific iodine potassium iodide, and eosin Y, which mostly interacts with proteins and peptides in the cell cytoplasm, the distribution of the stains within the cells in murine kidney samples is assessed and compared to unstained samples with similar structural features. Quantitative nanoscopic stain concentrations are in good agreement with dual-energy micro computed tomography measurements, the state-of-the-art modality for material-selective imaging. The presented approach can be applied to a variety of X-ray stains advancing the development of X-ray contrast agents.

Model-Based Iterative Reconstruction for Propagation-Based Phase-Contrast X-Ray CT including Models for the Source and the Detector.

Propagation-based phase-contrast X-ray computed tomography is a valuable tool for high-resolution visualization of biological samples, giving distinct improvements in terms of contrast and dose requirements compared to conventional attenuation-based computed tomography. Due to its ease of implementation and advances in laboratory X-ray sources, this imaging technique is increasingly being transferred from synchrotron facilities to laboratory environments. This however poses additional challenges, such as the limited spatial coherence and flux of laboratory sources, resulting in worse resolution and higher noise levels. Here we extend a previously developed iterative reconstruction algorithm for this imaging technique to include models for the reduced spatial coherence and the signal spreading of efficient scintillator-based detectors directly into the physical forward model. Furthermore, we employ a noise model which accounts for the full covariance statistics of the image formation process. In addition, we extend the model describing the interference effects such that it now matches the formalism of the widely-used single-material phase-retrieval algorithm, which is based on the sample-homogeneity assumption. We perform a simulation study as well as an experimental study at a laboratory inverse Compton source and compare our approach to the conventional analytical approaches. We find that the modeling of the source and the detector inside the physical forward model can tremendously improve the resolution at matched noise levels and that the modeling of the covariance statistics reduces overshoots (i.e. incorrect increase / decrease in sample properties) at the sample edges significantly.

Energy-Dispersive X-ray Absorption Spectroscopy with an Inverse Compton Source.

Novel compact x-ray sources based on inverse Compton scattering can generate brilliant hard x-rays in a laboratory setting. Their collimated intense beams with tunable well-defined x-ray energies make them well suited for x-ray spectroscopy techniques, which are typically carried out at large facilities. Here, we demonstrate a first x-ray absorption spectroscopy proof-of-principle experiment using an inverse Compton x-ray source with a flux of >1010 photons/s in <5% bandwidth. We measured x-ray absorption near edge structure and extended x-ray absorption fine structure at the silver K-edge (~25.5 keV) for a series of silver samples. We propose an energy-dispersive geometry specifically adapted to the x-ray beam properties of inverse Compton x-ray sources together with a fast concentration correction method that corrects sample inhomogeneities very effectively. The combination of our setup with the inverse Compton source generates x-ray absorption spectra with high energy resolution in exposure times down to one minute. Our results unravel the great benefit of inverse Compton scattering sources for x-ray absorption techniques in a laboratory environment, especially in the hard x-ray regime, which allows to probe absorption edges of high Z materials.

Spectroscopic imaging at compact inverse Compton X-ray sources.

While K-edge subtraction (KES) imaging is a commonly applied technique at synchrotron sources, the application of this imaging method in clinical imaging is limited although results have shown its superiority to conventional clinical subtraction imaging. Over the past decades, compact synchrotron X-ray sources, based on inverse Compton scattering, have been developed to fill the gap between conventional X-ray tubes and synchrotron facilities. These so called inverse Compton sources (ICSs) provide a tunable, quasi-monochromatic X-ray beam in a laboratory setting with reduced spatial and financial requirements. This allows for the transfer of imaging techniques that have been limited to synchrotrons until now, like KES imaging, into a laboratory environment. This review article presents the first studies that have successfully performed KES at ICSs. These have shown that KES provides improved image quality in comparison to conventional X-ray imaging. The results indicate that medical imaging could benefit from monochromatic imaging and KES techniques. Currently, the clinical application of KES is limited by the low K-edge energy of available iodine contrast agents. However, several ICSs are under development or already in commissioning which will provide monochromatic X-ray beams with higher X-ray energies and will enable KES using high-Z elements as contrast media. With these developments, KES at an ICS has the ability to become an important tool in pre-clinical research and potentially advancing existing clinical imaging techniques.

K-edge subtraction imaging for iodine and calcium separation at a compact synchrotron x-ray source.

Purpose: About one third of all deaths worldwide can be traced to some form of cardiovascular disease. The gold standard for the diagnosis and interventional treatment of blood vessels is digital subtraction angiography (DSA). An alternative to DSA is K-edge subtraction (KES) imaging, which has been shown to be advantageous for moving organs and for eliminating image artifacts caused by patient movement. As highly brilliant, monochromatic x-rays are required for this method, it has been limited to synchrotron facilities so far, restraining the applicability in the clinical routine. Over the past decades, compact synchrotron x-ray sources based on inverse Compton scattering have been evolving; these provide x-rays with sufficient brilliance and meet spatial and financial requirements for laboratory settings or university hospitals. Approach: We demonstrate a proof-of-principle KES imaging experiment using the Munich Compact Light Source (MuCLS), the first user-dedicated installation of a compact synchrotron x-ray source worldwide. A series of experiments were performed both on a phantom and an excised human carotid to demonstrate the ability of the proposed KES technique to separate the iodine contrast agent and calcifications. Results: It is shown that the proposed filter-based KES method allows for the iodine-contrast agent and calcium to be clearly separated, thereby providing x-ray images only showing one of the two materials. Conclusions: The results show that the quasimonochromatic spectrum of the MuCLS enables filter-based KES imaging and can become an important tool in preclinical research and possible future clinical diagnostics.