K-edge subtraction synchrotron X-ray imaging in bio-medical research

High contrast in X-ray medical imaging, while maintaining acceptable radiation dose levels to the patient, has long been a goal. One of the most promising methods is that of K-edge subtraction imaging. This technique, first advanced as long ago as 1953 by B. Jacobson, uses the large difference in the absorption coefficient of elements at energies above and below the K-edge. Two images, one taken above the edge and one below the edge, are subtracted leaving, ideally, only the image of the distribution of the target element. This paper reviews the development of the KES techniques and technology as applied to bio-medical imaging from the early low-power tube sources of X-rays to the latest high-power synchrotron sources. Applications to coronary angiography, functional lung imaging and bone growth are highlighted. A vision of possible imaging with new compact sources is presented.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

BMIT facility at the Canadian Light Source: Advances in X-ray phase-sensitive imaging

The BioMedical Imaging and Therapy (BMIT) facility [1,2] located at the Canadian Light Source, provides synchrotron-specific imaging and radiation therapy capabilities. There are two separate beamlines used for experiments: the bending magnet (05B1-1) and the insertion device (05ID-2) beamline.The bending magnet beamline provides access to monochromatic beam spanning a spectral range of 15–40 keV, and the beam is 240 mm wide in the POE-2 experimental hutch. Users can also perform experiments with polychromatic (pink) beam.The insertion device beamline was officially opened for general user program in 2015. The source for the ID beamline is a multi-pole, superconducting 4.3 T wiggler. The high field gives a critical energy over 20 keV. The optics hutches prepare a beam that is 220 mm wide in the last experimental hutch SOE-1. The monochromatic spectral range spans 25–150+ keV. Several different X-ray detectors are available for both beamlines, with resolutions ranging from 2 μm to 200 μm.BMIT provides a number of imaging techniques including standard absorption X-ray imaging, K-edge subtraction imaging (KES), in-line phase contrast imaging (also known as propagation based imaging, PBI) and Diffraction Enhanced Imaging/Analyzer Based Imaging (DEI/ABI), all in either projection or CT mode. PBI and DEI/ABI are particularly important tools for BMIT users since these techniques enable visualization of soft tissue and allow for low dose imaging.     

Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.     

Synchrotron based planar imaging and digital tomosynthesis of breast and biopsy phantoms using a CMOS active pixel sensor

The SYRMEP (SYnchrotron Radiation for MEdical Physics) beamline at Elettra is performing the first mammography study on human patients using free-space propagation phase contrast imaging. The stricter spatial resolution requirements of this method currently force the use of conventional films or specialized computed radiography (CR) systems. This also prevents the implementation of three-dimensional (3D) approaches. This paper explores the use of an X-ray detector based on complementary metal-oxide-semiconductor (CMOS) active pixel sensor (APS) technology as a possible alternative, for acquisitions both in planar and tomosynthesis geometry.Results indicate higher quality of the images acquired with the synchrotron set-up in both geometries. This improvement can be partly ascribed to the use of parallel, collimated and monochromatic synchrotron radiation (resulting in scatter rejection, no penumbra-induced blurring and optimized X-ray energy), and partly to phase contrast effects. Even though the pixel size of the used detector is still too large – and thus suboptimal – for free-space propagation phase contrast imaging, a degree of phase-induced edge enhancement can clearly be observed in the images.     

Potential of compact Compton sources in the medical field

The exceptional improvement of high power lasers and optical cavity finesses in the last fifteen years allows today the development of X-ray sources based on inverse Compton scattering. These compact sources will provide high intensity beams, with a tunable energy in the range 20–100 keV, that can be used in several application including material sciences, structural biology, cultural heritage research and preservation and medical or biomedical preclinical and clinical research. The access to these devices will be easier. Methods currently used only in synchrotron facilities will be available in dedicated work environment such as hospitals, laboratories or museums. Several machines are in design or construction phase, and aim at producing 10¹²–10¹⁴ ph/s. The ThomX machine is the most advanced project and has the potential to be used as the radiation source for biomedical searches, clinical imaging techniques or radiotherapy programs.     

两种微体化石三维无损成像技术的对比

近年来,各种X射线三维无损成像技术在古生物学领域的应用越来越广泛。但是,不同的X射线三维无损成像技术针对不同保存类型和尺寸的化石标本在成像效果上各有利弊。本文以埃迪卡拉纪陡山沱组磷酸盐化的动物胚胎化石为研究对象,将目前应用最广的两种X射线三维无损成像方法,即基于实验室X光源的吸收衬度显微断层成像技术和基于同步辐射光源的相位衬度显微断层成像技术进行了对比分析。通过对两种技术的原理、效率、空间分辨率和图像衬度的对比,认为基于同步辐射光源的相位衬度显微断层成像技术是目前对于均一矿化的微体化石最佳的三维无损成像解决方案。     

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.     

Research at the European Synchrotron Radiation Facility medical beamline.

The application of synchrotron radiation in medical research has become a mature field of research at synchrotron facilities worldwide. In the relatively short time that synchrotrons have been available to the scientific community, their characteristic beams of UV and X-ray radiation have been applied to virtually all areas of medical science which use ionizing radiation. The ability to tune intense monochromatic beams over wide energy ranges differentiates these sources from standard clinical and research tools. At the European Synchrotron Radiation Facility (Grenoble, France), a major research facility is operational on an advanced wiggler radiation beamport, ID17. The beamport is designed to carry out a broad range of research ranging from cell radiation biology to in vivo human studies. Medical imaging programs at ID17 include transvenous coronary angiography, computed tomography, mammography and bronchography. In addition, a major research program on microbeam radiation therapy is progressing. This paper will present a very brief overview of the beamline and the imaging and therapy programs.     

Laboratory cryo soft X-ray microscopy

Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a λ=2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined.     

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult – if not impossible – to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

X-ray phase imaging reaching clinical uses

X-ray phase imaging that uses the phenomena of X-ray refraction and scattering to generate image contrast has the potential to overcome the drawback of conventional X-ray radiography in observing biological soft tissues. After its dawn at synchrotron radiation facilities 30 years ago, the development of X-ray phase imaging is expanding to hospitals by grating-based phase-imaging approaches available with a conventional X-ray tube. In this review, after introducing the physical advantages and methodological details of X-ray phase imaging, recent trials of instrumentation in hospitals for diagnoses of rheumatoid arthritis and chronic obstructive pulmonary disease are introduced.     

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI–CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later ‘stitched’ together, forming a 3D dataset. High-resolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities.Abbreviations: DEI, diffraction-enhanced imagingDiffraction-enhanced imaging (DEI) is a biomedical imaging technique that, compared with conventional radiography, generates very detailed images with more edge contrast but deposits a lower radiation dose to the object. DEI generates enhanced contrast both from absorption, the process involved in conventional radiography, and from of X-ray refraction, a process that harnesses photons that otherwise typically are imperceptibly diffracted.⁴ The DEI technique collects information from X-rays that are refracted as they pass through tissues that have different refractive indices as it almost completely removes diffracted X-rays. In comparison, conventional radiography produces images from X-rays that are attenuated by the tissues through which they pass, but X-rays that are refracted within those same tissues confound, rather than clarify, image contrast. The creation of contrast from the refraction of X-rays, rather than exclusively from absorption, yields images that display more detail with clearer distinction between tissue interfaces. Refraction-based imaging can reveal tiny structures that are transparent to X-ray attenuation but have sufficient variation in density to produce refraction contrast. Furthermore, refraction-based imaging decreases the required radiation dose.²¹To obviate the superimposing effects in a 2-dimensional DEI refraction image, we considered that combining CT with DEI would yield images with even greater clarity. CT allows a 3D representation of the sample, such that contrast from features at different depths are no longer superimposed on one another but can be separated and viewed as independent structures. Although this advantage is valuable in traditional absorption imaging, the additional features that provide contrast in a refraction-based image enhance the value of CT. Combining DEI technology, which is capable of imaging soft-tissue detail, with CT, which allows segregation of the contrast images at different depths, overcomes limitations of conventional X-ray imaging, namely lack of distinction of soft tissues and 2-dimensionality. As we report here, DEI combined with CT and a synchrotron-generated X-ray source yields 3D images of growing joint tissues at a resolution on the order of micrometers, which is much higher than can be generated using conventional imaging techniques.A synchrotron radiation source was required for the development of DEI because a synchrotron currently is the only source capable of providing an intensely brilliant light (millions of times brighter than sunlight and conventional X-ray sources), is highly collimated (light rays in the beam remain parallel with negligible dispersion over distance), can be made to be monochromatic (having a single wavelength), and can be tuned precisely to an array of energy ranges. The Canadian Light Source (www.lightsource.ca), which began operations in 2005, is one of only 47 synchrotron facilities worldwide and the only such facility in Canada. Although nonsynchrotron sources of X-rays for DEI–CT are conceivable,^16,18 such technology requires considerable image-acquisition time. Regardless, the quality of images generated by using synchrotron technology likely would remain the standard with which any new nonsynchrotron DEI–CT technological innovations would be compared.¹⁴Despite refinements in medical imaging, conventional radiography, CT scanning, and MRI still are insufficient to discern fine details, particularly in growing joints in which soft tissues (including cartilage) predominate and change with physiologic growth. The impetus for the current research was to develop an imaging technique that better demonstrated normal joint characteristics during growth and, in the future, could be applied to pathologic joints for experimental research and eventually clinical applications. In particular, we were motivated by a need to more effectively and reliably image growing joints affected by arthritis, a disease associated with alterations of bone and cartilage growth, tissue morphology and vascularity. Childhood arthritis research likely will benefit from having an improved imaging technique to aid in early diagnosis, monitor disease progression, and assess responses to therapies. The long-term outcomes of childhood arthritis are improved with early diagnosis and prompt and effective response to treatment interventions. Clinical and laboratory-based indicators of inflammation are not always adequate to detect and monitor subclinical intraarticular inflammation which, as with overt disease, can lead to progressive joint damage. Imaging can augment clinical and laboratory assessment of arthritis activity, but even the most sensitive currently available modalities are unable to detect all joint pathology.In juvenile arthritis, joint-imaging outcomes are difficult to evaluate because variations associated with normal growth cannot always be easily discerned from variations induced by the disease. Conventional radiography tends to detect advanced joint damage that has affected bone, but cartilage can be assessed only indirectly, and soft tissue abnormalities cannot be fully evaluated. Consequently, conventional radiography has insufficient sensitivity and specificity to be considered useful for diagnosing or monitoring children with inflammatory joint disease.^6,20 MRI, which evaluates both soft tissues and osteochondral structures, can be used to detect cartilage loss, bone erosions, and synovial hypertrophy in children and adolescents, and contrast-enhanced MRI detects active synovitis.^1,10 However, standardized approaches to acquire and interpret MRI data are not established for children in general and, in particular, for children with arthritis;^12,15 it is not always clear, for example, if observed thinning of cartilage is physiologic or pathologic. Furthermore, although MRI is more sensitive than conventional radiography, MRI too has limited precision in detecting fine structures and pathologic changes; a clinical MRI has less than 50% sensitivity in detecting cartilage damage that subsequently is seen arthroscopically.^8,13CT offers another option for joint visualization, given that it provides high-resolution, 3D images of bone from any angle. Despite its high spatial resolution, however, CT cannot match MRI''s soft-tissue contrast resolution, because CT provides negligible variability of attenuation coefficients of soft tissues so attenuation is nearly the same for cartilage, muscles, and ligaments. Furthermore, CT''s value is offset by the necessity for radiation exposure, a particular concern in the pediatric population. Therefore, for joint research and clinical applications, each of the conventional imaging techniques currently available has limitations. A safe, higher resolution imaging system that generates good contrast for all joint structures is required.Because the DEI technique initially was developed by using a synchrotron light source, we similarly used synchrotron technology in the current experiments. In contrast to conventional X-ray tubes, a synchrotron generates light by using radiofrequency waves and electromagnets to energize and accelerate electrons, thus producing brilliant, highly focused light from the entire wavelength spectrum, including X-rays. For the development and evaluation of DEI–CT imaging of joints, we chose to use healthy commercial piglet stifle joints because porcine stifle joints are anatomically similar to human knees.⁵ In addition, pigs grow quickly, reaching skeletal maturity at the distal femur and proximal tibia in 20 mo,¹⁹ thus allowing for the use of the pig as a model to study growth patterns in normal and disease states in a relatively short time period. The current study aimed to develop and test a new technology for imaging growing joints by using DEI combined with CT and a synchrotron radiation source. This report is the first to document the application of DEI–CT for imaging intact, growing joints.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

多功能磁共振造影剂研究进展

磁共振成像技术因对人体无创、任意方向断层扫描三维图像且分辨率较高、提供形态与功能两方面诊断评价等突出优点,成为了临床上用于疾病诊断的重要手段之一。临床上使用磁共振造影剂可以提高成像的分辨率和灵敏度,提高图像质量,增强对比度和可读性。但是,各种成像技术由于实现原理不同,具有各自的优势和缺陷,靠传统单一的诊断模式无法提供疾病的全面信息,因而在对各种复杂疾病进行诊断时会受到一定的限制。因此,将磁共振成像与其他成像技术如CT成像、超声成像等联合起来使用,则可以达到优势互补的效果,能为疾病的临床诊断提供更快捷精确的信息,同时可将磁共振成像与各种治疗方式结合在一起,即开发基于磁共振成像的诊断治疗一体化试剂,以实现对疾病的即时治疗和实时监控。本文主要介绍了磁共振成像造影剂的原理和种类,并且综述了目前国内外在基于磁共振成像的多功能造影剂/诊疗制剂这一领域的研究进展,最后就未来可能的研究方向进行了展望。     

Protein crystallography microdiffraction

Protein microdiffraction using monochromatic beams is becoming a routine tool at third-generation synchrotron radiation sources. Beam sizes have reached the scale of about 5 microm, with illuminated crystal volumes of approximately 500 microm3, as shown for the case of bovine rhodopsin, which was refined to a resolution of 2.6 A. Progress in X-ray optical systems and instrumentation will enable the method to be extended to smaller beams and smaller crystal volumes.     

A single-image retrieval method for edge illumination X-ray phase-contrast imaging: Application and noise analysis

PurposeEdge illumination (EI) X-ray phase-contrast imaging (XPCI) has been under development at University College London in recent years, and has shown great potential for both laboratory and synchrotron applications. In this work, we propose a new acquisition and processing scheme. Contrary to existing retrieval methods for EI, which require as input two images acquired in different setup configurations, the proposed approach can retrieve an approximate map of the X-ray phase from a single image, thus significantly simplifying the acquisition procedure and reducing data collection times.MethodsThe retrieval method is analytically derived, based on the assumption of a quasi-homogeneous object, i.e. an object featuring a constant ratio between refractive index and absorption coefficient. The noise properties of the input and retrieved images are also theoretically analyzed under the developed formalism. The method is applied to experimental synchrotron images of a biological object.ResultsThe experimental results show that the method can provide high-quality images, where the “edge” signal typical of XPCI images is transformed to an “area” contrast that enables an easier interpretation of the sample geometry. Moreover, the retrieved images confirm that the method is highly stable against noise.ConclusionsWe anticipate that the developed approach will become the method of choice for a variety of applications of EI XPCI, thanks to its ability to simplify the acquisition procedure and reduce acquisitions time and dose to the sample. Future work will focus on the adaptation of the method to computed tomography and to polychromatic radiation from X-ray tubes.     

High quality 3D imaging of vertebrate microremains using X-ray synchrotron phase contrast microtomography

Vertebrate microremains, particularly teeth, represent a substantial part of known vertebrate biodiversity. Many groups, such as Mesozoic mammals, are known mostly through isolated teeth. Classical imaging techniques of such complex millimetric to inframillime-tric objects are most often limited by problems of manipulation, depth of focus or limited orientation. The methods generally used are stereomicroscopy (including in-focus z-series reconstruction) and Scanning Electron Microscopy (SEM), which provide good images. Nevertheless, both provide 2D static images or partial and directional 3D data, making complete observation difficult. Propagation phase contrast synchrotron X-ray microtomography is a powerful technique alleviating these limitations. Thanks to submicron resolution and to the edge detection effect, it rapidly provides 3D data from minute samples with levels of quality and detail unattainable using conventional microtomographs. Complex morphology of small specimens can be studied with unlimited orientation possibilities and, when coupled with 3D printing, it allows enlarged 3D reproductions of such small and fragile fossils.