Detection of circulating tumor cells via an X‐ray imaging technique

Detailed information on the location and the size of tumor cells circulating through lymphatic and blood vessels is useful to cancer diagnosis. Metastasis of cancers to other non‐adjacent organs is reported to cause 90% of deaths not from the primary tumors. Therefore, effective detection of circulating tumors cells (CTCs) related to metastasis is emphasized in cancer treatments. With the use of synchrotron X‐ray micro‐imaging techniques, high‐resolution images of individual flowing tumor cells were obtained. Positively charged gold nanoparticles (AuNPs) which were inappropriate for incorporation into human red blood cells were selectively incorporated into tumor cells to enhance the image contrast. This approach enables images of individual cancer cells and temporal movements of CTCs to be captured by the high X‐ray absorption efficiency of selectively incorporated AuNPs. This new technology for in vivo imaging of CTCs would contribute to improve cancer diagnosis and cancer therapy prognosis.     

In vivo high‐resolution synchrotron radiation imaging of collagen‐induced arthritis in a rodent model

In vivo microstructures of the affected feet of collagen‐induced arthritic (CIA) mice were examined using a high‐resolution synchrotron radiation (SR) X‐ray refraction technique with a polychromatic beam issued from a bending magnet. The CIA models were obtained from six‐week‐old DBA/1J mice that were immunized with bovine type II collagen and grouped as grades 0–3 according to a clinical scoring for the severity of arthritis. An X‐ray shadow of a specimen was converted into a visual image on the surface of a CdWO₄ scintillator that was magnified using a microscopic objective lens before being captured with a digital charge‐coupled‐device camera. Various changes in the joint microstructure, including cartilage destruction, periosteal born formation, articular bone thinning and erosion, marrow invasion by pannus progression, and widening joint space, were clearly identified at each level of arthritis severity with an equivalent pixel size of 2.7 µm. These high‐resolution features of destruction in the CIA models have not previously been available from any other conventional imaging modalities except histological light microscopy. However, thickening of the synovial membrane was not resolved in composite images by the SR refraction imaging method. In conclusion, in vivo SR X‐ray microscopic imaging may have potential as a diagnostic tool in small animals that does not require a histochemical preparation stage in examining microstructural changes in joints affected with arthritis. The findings from the SR images are comparable with standard histopathology findings.     

Potential of propagation‐based synchrotron X‐ray phase‐contrast computed tomography for cardiac tissue engineering

Hydrogel‐based cardiac tissue engineering offers great promise for myocardial infarction repair. The ability to visualize engineered systems in vivo in animal models is desired to monitor the performance of cardiac constructs. However, due to the low density and weak X‐ray attenuation of hydrogels, conventional radiography and micro‐computed tomography are unable to visualize the hydrogel cardiac constructs upon their implantation, thus limiting their use in animal systems. This paper presents a study on the optimization of synchrotron X‐ray propagation‐based phase‐contrast imaging computed tomography (PCI‐CT) for three‐dimensional (3D) visualization and assessment of the hydrogel cardiac patches. First, alginate hydrogel was 3D‐printed into cardiac patches, with the pores filled by fibrin. The hydrogel patches were then surgically implanted on rat hearts. A week after surgery, the hearts including patches were excised and embedded in a soft‐tissue‐mimicking gel for imaging by using PCI‐CT at an X‐ray energy of 25 keV. During imaging, the sample‐to‐detector distances, CT‐scan time and the region of interest (ROI) were varied and examined for their effects on both imaging quality and radiation dose. The results showed that phase‐retrieved PCI‐CT images provided edge‐enhancement fringes at a sample‐to‐detector distance of 147 cm that enabled visualization of anatomical and microstructural features of the myocardium and the implanted patch in the tissue‐mimicking gel. For visualization of these features, PCI‐CT offered a significantly higher performance than the dual absorption‐phase and clinical magnetic resonance (3 T) imaging techniques. Furthermore, by reducing the total CT‐scan time and ROI, PCI‐CT was examined for lowering the effective dose, meanwhile without much loss of imaging quality. In effect, the higher soft tissue contrast and low‐dose potential of PCI‐CT has been used along with an acceptable overall animal dose to achieve the high spatial resolution needed for cardiac implant visualization. As a result, PCI‐CT at the identified imaging parameters offers great potential for 3D assessment of microstructural features of hydrogel cardiac patches.     

Comparison of in vitro breast cancer visibility in analyser‐based computed tomography with histopathology,mammography, computed tomography and magnetic resonance imaging

High‐resolution analyser‐based X‐ray imaging computed tomography (HR ABI‐CT) findings on in vitro human breast cancer are compared with histopathology, mammography, computed tomography (CT) and magnetic resonance imaging. The HR ABI‐CT images provided significantly better low‐contrast visibility compared with the standard radiological images. Fine cancer structures indistinguishable and superimposed in mammograms were seen, and could be matched with the histopathological results. The mean glandular dose was less than 1 mGy in mammography and 12–13 mGy in CT and ABI‐CT. The excellent visibility of in vitro breast cancer suggests that HR ABI‐CT may have a valuable role in the future as an adjunct or even alternative to current breast diagnostics, when radiation dose is further decreased, and compact synchrotron radiation sources become available.     

Design of a mouse restraint for synchrotron‐based computed tomography imaging

High‐resolution computed tomography (CT) imaging of a live animal within a lead‐lined synchrotron light hutch presents several unique challenges. In order to confirm that the animal is under a stable plane of anaesthesia, several physiological parameters (e.g. heart rate, arterial oxygen saturation, core body temperature and respiratory rate) must be remotely monitored from outside the imaging hutch. In addition, to properly scan the thoracic region using CT, the animal needs to be held in a vertical position perpendicular to the fixed angle of the X‐ray beam and free to rotate 180°–360°. A new X‐ray transparent mouse restraint designed and fabricated using computer‐aided design software and three‐dimensional rapid prototype printing has been successfully tested at the Biomedical Imaging and Therapy bending‐magnet (BMIT‐BM) beamline at the Canadian Light Source.     

Feasibility study of propagation‐based phase‐contrast X‐ray lung imaging on the Imaging and Medical beamline at the Australian Synchrotron

Propagation‐based phase‐contrast X‐ray imaging (PB‐PCXI) using synchrotron radiation has achieved high‐resolution imaging of the lungs of small animals both in real time and in vivo. Current studies are applying such imaging techniques to lung disease models to aid in diagnosis and treatment development. At the Australian Synchrotron, the Imaging and Medical beamline (IMBL) is well equipped for PB‐PCXI, combining high flux and coherence with a beam size sufficient to image large animals, such as sheep, due to a wiggler source and source‐to‐sample distances of over 137 m. This study aimed to measure the capabilities of PB‐PCXI on IMBL for imaging small animal lungs to study lung disease. The feasibility of combining this technique with computed tomography for three‐dimensional imaging and X‐ray velocimetry for studies of airflow and non‐invasive lung function testing was also investigated. Detailed analysis of the role of the effective source size and sample‐to‐detector distance on lung image contrast was undertaken as well as phase retrieval for sample volume analysis. Results showed that PB‐PCXI of lung phantoms and mouse lungs produced high‐contrast images, with successful computed tomography and velocimetry also being carried out, suggesting that live animal lung imaging will also be feasible at the IMBL.     

Design,development and first experiments on the X‐ray imaging beamline at Indus‐2 synchrotron source RRCAT,India

A full‐field hard X‐ray imaging beamline (BL‐4) was designed, developed, installed and commissioned recently at the Indus‐2 synchrotron radiation source at RRCAT, Indore, India. The bending‐magnet beamline is operated in monochromatic and white beam mode. A variety of imaging techniques are implemented such as high‐resolution radiography, propagation‐ and analyzer‐based phase contrast imaging, real‐time imaging, absorption and phase contrast tomography etc. First experiments on propagation‐based phase contrast imaging and micro‐tomography are reported.     

Early commissioning results for spectroscopic X‐ray Nano‐Imaging Beamline BL 7C sXNI at PLS‐II

For spectral imaging of chemical distributions using X‐ray absorption near‐edge structure (XANES) spectra, a modified double‐crystal monochromator, a focusing plane mirrors system and a newly developed fluorescence‐type X‐ray beam‐position monitoring and feedback system have been implemented. This major hardware upgrade provides a sufficiently stable X‐ray source during energy scanning of more than hundreds of eV for acquisition of reliable XANES spectra in two‐dimensional and three‐dimensional images. In recent pilot studies discussed in this paper, heavy‐metal uptake by plant roots in vivo and iron's phase distribution in the lithium–iron–phosphate cathode of a lithium‐ion battery have been imaged. Also, the spatial resolution of computed tomography has been improved from 70 nm to 55 nm by means of run‐out correction and application of a reconstruction algorithm.     

Live small‐animal X‐ray lung velocimetry and lung micro‐tomography at the Australian Synchrotron Imaging and Medical Beamline

The high flux and coherence produced at long synchrotron beamlines makes them well suited to performing phase‐contrast X‐ray imaging of the airways and lungs of live small animals. Here, findings of the first live‐animal imaging on the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron are reported, demonstrating the feasibility of performing dynamic lung motion measurement and high‐resolution micro‐tomography. Live anaesthetized mice were imaged using 30 keV monochromatic X‐rays at a range of sample‐to‐detector propagation distances. A frame rate of 100 frames s^?1 allowed lung motion to be determined using X‐ray velocimetry. A separate group of humanely killed mice and rats were imaged by computed tomography at high resolution. Images were reconstructed and rendered to demonstrate the capacity for detailed, user‐directed display of relevant respiratory anatomy. The ability to perform X‐ray velocimetry on live mice at the IMBL was successfully demonstrated. High‐quality renderings of the head and lungs visualized both large structures and fine details of the nasal and respiratory anatomy. The effect of sample‐to‐detector propagation distance on contrast and resolution was also investigated, demonstrating that soft tissue contrast increases, and resolution decreases, with increasing propagation distance. This new capability to perform live‐animal imaging and high‐resolution micro‐tomography at the IMBL enhances the capability for investigation of respiratory diseases and the acceleration of treatment development in Australia.     

X‐ray microtomographic visualization of Escherichia coli by metalloprotein overexpression

This paper reports X‐ray microtomographic visualization of the microorganism Escherichia coli overexpressing a metalloprotein ferritin. The three‐dimensional distribution of linear absorption coefficients determined using a synchrotron radiation microtomograph with a simple projection geometry revealed that the X‐ray absorption was homogeneously distributed, suggesting that every E. coli cell was labeled with the ferritin. The ferritin‐expressing E. coli exhibited linear absorption coefficients comparable with those of phosphotungstic acid stained cells. The submicrometer structure of the ferritin‐expressing E. coli cells was visualized by Zernike phase contrast using an imaging microtomograph equipped with a Fresnel zone plate. The obtained images revealed curved columnar or bunching oval structures corresponding to the E. coli cells. These results indicate that the metalloprotein overexpression facilitates X‐ray visualization of three‐dimensional cellular structures of biological objects.     

A new approach to synchrotron energy‐dispersive X‐ray diffraction computed tomography

A new data collection strategy for performing synchrotron energy‐dispersive X‐ray diffraction computed tomography has been devised. This method is analogous to angle‐dispersive X‐ray diffraction whose diffraction signal originates from a line formed by intersection of the incident X‐ray beam and the sample. Energy resolution is preserved by using a collimator which defines a small sampling voxel. This voxel is translated in a series of parallel straight lines covering the whole sample and the operation is repeated at different rotation angles, thus generating one diffraction pattern per translation and rotation step. The method has been tested by imaging a specially designed phantom object, devised to be a demanding validator for X‐ray diffraction imaging. The relative strengths and weaknesses of the method have been analysed with respect to the classic angle‐dispersive technique. The reconstruction accuracy of the method is good, although an absorption correction is required for lower energy diffraction because of the large path lengths involved. The spatial resolution is only limited to the width of the scanning beam owing to the novel collection strategy. The current temporal resolution is poor, with a scan taking several hours. The method is best suited to studying large objects (e.g. for engineering and materials science applications) because it does not suffer from diffraction peak broadening effects irrespective of the sample size, in contrast to the angle‐dispersive case.     

High‐resolution thermal imaging with a combination of nano‐focus X‐ray diffraction and ultra‐fast chip calorimetry

A microelectromechanical‐systems‐based calorimeter designed for use on a synchrotron nano‐focused X‐ray beamline is described. This instrument allows quantitative DC and AC calorimetric measurements over a broad range of heating/cooling rates (≤100000 K s^?1) and temperature modulation frequencies (≤1 kHz). The calorimeter was used for high‐resolution thermal imaging of nanogram‐sized samples subjected to X‐ray‐induced heating. For a 46 ng indium particle, the measured temperature rise reaches ～0.2 K, and is directly correlated to the X‐ray absorption. Thermal imaging can be useful for studies of heterogeneous materials exhibiting physical and/or chemical transformations. Moreover, the technique can be extended to three‐dimensional thermal nanotomography.     

X‐ray fluorescent CT imaging of cerebral uptake of stable‐iodine perfusion agent iodoamphetamine analog IMP in mice

Using X‐ray fluorescent computed tomography (XFCT), the in vivo and ex vivo cerebral distribution of a stable‐iodine‐labeled cerebral perfusion agent, iodoamphetamine analog (¹²⁷I‐IMP), has been recorded in the brains of mice. In vivo cerebral perfusion in the cortex, hippocampus and thalamus was depicted at 0.5 mm in‐plane spatial resolution. Ex vivo XFCT images at 0.25 mm in‐plane spatial resolution allowed the visualisation of the detailed structures of these regions. The quality of the XFCT image of the hippocampus was comparable with the ¹²⁵I‐IMP autoradiogram. These results highlight the sensitivity of XFCT and its considerable potential to evaluate cerebral perfusion in small animals without using radioactive agents.     

Methodological challenges of optical tweezers‐based X‐ray fluorescence imaging of biological model organisms at synchrotron facilities

Recently, a radically new synchrotron radiation‐based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non‐contact laser‐based sample manipulation with synchrotron radiation confocal X‐ray fluorescence (XRF) microimaging for the first time at ESRF‐ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF‐related challenges are reported. In general, the applicability of the OT‐based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi‐elemental analysis is of relevance at the (sub)micrometre spatial resolution level.     

First experimental result with fluorescent X‐ray CT based on sheet‐beam geometry

Fluorescent X‐ray computed tomography (FXCT) enables us to reveal the cross‐sectional distribution of very low concentration of specific elements, e.g. I, Gd, or Au, in biomedical samples at a spatial resolution of several hundred micrometers, and it has been used to evaluate the states of cerebral perfusion and fatty acid metabolic function of small animals in vivo and ex vivo. However, since the current system employs the data‐acquisition scheme of the first‐generation type of CT, it requires a huge amount of time to obtain a whole set of projections. In order to overcome the problem, we propose a novel imaging geometry using a sheet incident beam. We performed a proof‐of‐concept experiment using a preliminary imaging system constructed at beam line BLNE‐5A, KEK. The efficacy of the proposed method is demonstrated by the reconstructed images of a physical phantom containing various concentrations of iodine solution and a mouse brain that is extracted after intravenous injection of ¹²⁷I‐IMP for observing the cerebral perfusion and then fixed with formalin. Copyright © 2009 John Wiley & Sons, Ltd.     

Element‐specific microtomographic imaging of Drosophila brain stained with high‐Z probes

An application of X‐ray microtomography to the Drosophila adult brain stained with colloidal gold and a platinum compound is described. The transparency of biological tissue to hard X‐rays enables tomographic visualization of the three‐dimensional structure of tissue entrails. Each high‐Z element was visualized as a three‐dimensional structure from the difference absorption coefficient image at the corresponding L_III absorption edge. The cortex of the optic lobe was selectively visualized by the specific adsorption of colloidal gold. The entire structure revealed by the platinum impregnation allowed the anatomical assignment of the gold‐stained structures. Selective staining and specific visualization of biological tissues at micrometer resolution should elucidate the three‐dimensional cellular organization essential for the understanding and application of biological microstructures.     

Radiobiological features of the anti‐cancer strategies involving synchrotron X‐rays

Synchrotrons are opening new paths in innovative anti‐cancer radiotherapy strategies. Indeed, the fluence of X‐rays induced by synchrotrons is so high (10⁶ times higher than standard medical irradiators) that it enables the production of X‐ray beams tunable in energy (monochromatic beams) and in size (micrometric beams). Monochromatic synchrotron X‐ray beams theoretically permit photoactivate high‐Z elements to be introduced in or close to tumours in order to increase the yield of damage by enhanced energy photoabsorption. This is notably the case of attempts with iodinated contrast agents used in tumour imaging (the computed tomography therapy approach) and with platinated agents used in chemotherapy (the PAT‐Plat approach). Micrometric synchrotron X‐ray beams theoretically permit very high radiation doses to accumulate in tumours by using arrays of parallel microplanar beams that spare the surrounding tissues (the microbeam radiation therapy approach). These anti‐cancer applications of synchrotron radiation have been developed at the European Synchrotron Radiation Facility to be applied to glioma, one of the tumour tissues most refractory to standard treatments. In the present paper the molecular and cellular mechanisms involved in these three approaches are reviewed, in the context of recent advances in radiobiology. Furthermore, by considering the unavoidable biases, an attempt to propose a comparison of the different results obtained in preclinical trials dealing with rats bearing tumours is given.     

A novel epitaxially grown LSO‐based thin‐film scintillator for micro‐imaging using hard synchrotron radiation

The efficiency of high‐resolution pixel detectors for hard X‐rays is nowadays one of the major criteria which drives the feasibility of imaging experiments and in general the performance of an experimental station for synchrotron‐based microtomography and radiography. Here the luminescent screen used for the indirect detection is focused on in order to increase the detective quantum efficiency: a novel scintillator based on doped Lu₂SiO₅ (LSO), epitaxially grown as thin film via the liquid phase epitaxy technique. It is shown that, by using adapted growth and doping parameters as well as a dedicated substrate, the scintillation behaviour of a LSO‐based thin crystal together with the high stopping power of the material allows for high‐performance indirect X‐ray detection. In detail, the conversion efficiency, the radioluminescence spectra, the optical absorption spectra under UV/visible‐light and the afterglow are investigated. A set‐up to study the effect of the thin‐film scintillator's temperature on its conversion efficiency is described as well. It delivers knowledge which is important when working with higher photon flux densities and the corresponding high heat load on the material. Additionally, X‐ray imaging systems based on different diffraction‐limited visible‐light optics and CCD cameras using among others LSO‐based thin film are compared. Finally, the performance of the LSO thin film is illustrated by imaging a honey bee leg, demonstrating the value of efficient high‐resolution computed tomography for life sciences.     

Hard X‐ray nanotomography beamline 7C XNI at PLS‐II

The synchrotron‐based hard X‐ray nanotomography beamline, named 7C X‐ray Nano Imaging (XNI), was recently established at Pohang Light Source II. This beamline was constructed primarily for full‐field imaging of the inner structures of biological and material samples. The beamline normally provides 46 nm resolution for still images and 100 nm resolution for tomographic images, with a 40 µm field of view. Additionally, for large‐scale application, it is capable of a 110 µm field of view with an intermediate resolution.     

Feasibility of in‐line instruments for high‐resolution inelastic X‐ray scattering

Inelastic X‐ray scattering instruments in operation at third‐generation synchrotron radiation facilities are based on backreflections from perfect silicon crystals. This concept reaches back to the very beginnings of high‐energy‐resolution X‐ray spectroscopy and has several advantages but also some inherent drawbacks. In this paper an alternate path is investigated using a different concept, the `M⁴ instrument'. It consists of a combination of two in‐line high‐resolution monochromators, focusing mirrors and collimating mirrors. Design choices and performance estimates in comparison with existing conventional inelastic X‐ray scattering instruments are presented.