Hard X‐ray phase‐contrast tomography of non‐homogeneous specimens: grating interferometry versus propagation‐based imaging

X‐ray phase‐contrast imaging is an effective approach to drastically increase the contrast and sensitivity of microtomographic techniques. Numerous approaches to depict the real part of the complex‐valued refractive index of a specimen are nowadays available. A comparative study using experimental data from grating‐based interferometry and propagation‐based phase contrast combined with single‐distance phase retrieval applied to a non‐homogeneous sample is presented (acquired at beamline ID19‐ESRF). It is shown that grating‐based interferometry can handle density gradients in a superior manner. The study underlines the complementarity of the two techniques for practical applications.     

Hard X‐ray phase‐contrast imaging with the Compact Light Source based on inverse Compton X‐rays

The first imaging results obtained from a small‐size synchrotron are reported. The newly developed Compact Light Source produces inverse Compton X‐rays at the intersection point of the counter propagating laser and electron beam. The small size of the intersection point gives a highly coherent cone beam with a few milliradian angular divergence and a few percent energy spread. These specifications make the Compact Light Source ideal for a recently developed grating‐based differential phase‐contrast imaging method.     

X‐ray analyzer‐based phase‐contrast computed laminography

X‐ray analyzer‐based phase‐contrast imaging is combined with computed laminography for imaging regions of interest in laterally extended flat specimens of weak absorption contrast. The optics discussed here consist of an asymmetrically cut collimator crystal and a symmetrically cut analyzer crystal arranged in a nondispersive (+, ?) diffraction geometry. A generalized algorithm is given for calculating multi‐contrast (absorption, refraction and phase contrast) images of a sample. Basic formulae are also presented for laminographic reconstruction. The feasibility of the method discussed was verified at the vertical wiggler beamline BL‐14B of the Photon Factory. At a wavelength of 0.0733 nm, phase‐contrast sectional images of plastic beads were successfully obtained. Owing to strong circular artifacts caused by a sample holder, the field of view was limited to about 6 mm in diameter.     

Enhanced renal image contrast by ethanol fixation in phase‐contrast X‐ray computed tomography

Phase‐contrast X‐ray imaging using a crystal X‐ray interferometer can depict the fine structures of biological objects without the use of a contrast agent. To obtain higher image contrast, fixation techniques have been examined with 100% ethanol and the commonly used 10% formalin, since ethanol causes increased density differences against background due to its physical properties and greater dehydration of soft tissue. Histological comparison was also performed. A phase‐contrast X‐ray system was used, fitted with a two‐crystal X‐ray interferometer at 35 keV X‐ray energy. Fine structures, including cortex, tubules in the medulla, and the vessels of ethanol‐fixed kidney could be visualized more clearly than that of formalin‐fixed tissues. In the optical microscopic images, shrinkage of soft tissue and decreased luminal space were observed in ethanol‐fixed kidney; and this change was significantly shown in the cortex and outer stripe of the outer medulla. The ethanol fixation technique enhances image contrast by approximately 2.7–3.2 times in the cortex and the outer stripe of the outer medulla; the effect of shrinkage and the physical effect of ethanol cause an increment of approximately 78% and 22%, respectively. Thus, the ethanol‐fixation technique enables the image contrast to be enhanced in phase‐contrast X‐ray imaging.     

Quantitative phase retrieval in X‐ray Zernike phase contrast microscopy

In recent years, increasing attention has been devoted to X‐ray phase contrast imaging, since it can provide high‐contrast images by using phase variations. Among the different existing techniques, Zernike phase contrast microscopy is one of the most popular phase‐sensitive techniques for investigating the fine structure of the sample at high spatial resolution. In X‐ray Zernike phase contrast microscopy, the image contrast is indeed a mixture of absorption and phase contrast. Therefore, this technique just provides qualitative information on the object, which makes the interpretation of the image difficult. In this contribution, an approach is proposed for quantitative phase retrieval in X‐ray Zernike phase contrast microscopy. By shifting the phase of the direct light by π/2 and 3π/2, two images of the same object are measured successively. The phase information of the object can then be quantitatively retrieved by a proper combination of the measured images. Numerical experiments were carried out and the results confirmed the feasibility of the proposed method. It is expected that the proposed method will find widespread applications in biology, materials science and so on.     

X‐ray grating interferometer for biomedical imaging applications at Shanghai Synchrotron Radiation Facility

An X‐ray grating interferometer was installed at the BL13W beamline of Shanghai Synchrotron Radiation Facility (SSRF) for biomedical imaging applications. Compared with imaging results from conventional absorption‐based micro‐computed tomography, this set‐up has shown much better soft tissue imaging capability. In particular, using the set‐up, the carotid artery and the carotid vein in a formalin‐fixed mouse can be visualized in situ without contrast agents, paving the way for future applications in cancer angiography studies. The overall results have demonstrated the broad prospects of the existing set‐up for biomedical imaging applications at SSRF.     

In vivo physiological saline‐infused hepatic vessel imaging using a two‐crystal‐interferometer‐based phase‐contrast X‐ray technique

Using a two‐crystal‐interferometer‐based phase‐contrast X‐ray imaging system, the portal vein, capillary vessel area and hepatic vein of live rats were revealed sequentially by injecting physiological saline via the portal vein. Vessels greater than 0.06 mm in diameter were clearly shown with low levels of X‐rays (552 µGy). This suggests that in vivo vessel imaging of small animals can be performed as conventional angiography without the side effects of the presently used iodine contrast agents.     

Advanced phase‐contrast imaging using a grating interferometer

Phase‐sensitive X‐ray imaging methods can provide substantially increased contrast over conventional absorption‐based imaging, and therefore new and otherwise inaccessible information. Differential phase‐contrast (DPC) imaging, which uses a grating interferometer and a phase‐stepping technique, has been integrated into TOMCAT, a beamline dedicated to tomographic microscopy and coherent radiology experiments at the Swiss Light Source. Developments have been made focusing on the fast acquisition and post‐processing of data to enable a high‐throughput of samples, with obvious advantages, also through increasing the efficiency of the detecting system, of helping to reduce radiation dose imparted to the sample. A novel aquarium design allows a vertical rotation axis below the sample with measurements performed in aqueous environment. Optimization of the data acquisition procedure enables a full phase volume (1024 × 1024 pixels × 1000 projections × 9 phase steps, i.e. 9000 projections in total) to be acquired in 20 min (with a pixel size of 7.4 µm), and the subsequent post‐processing has been integrated into the beamline pipeline for sinogram generation. Local DPC tomography allows one to focus with higher magnification on a particular region of interest of a sample without the presence of local tomography reconstruction artifacts. Furthermore, `widefield' imaging is shown for DPC scans for the first time, enabling the field of view of the imaging system to be doubled for samples that are larger than the magnification allows. A case study is illustrated focusing on the visualization of soft tissue features, and particularly the substantia nigra of a rat brain. Darkfield images, based on local X‐ray scattering, can also be extracted from a grating‐based DPC scan: an example of the advantages of darkfield contrast is shown and the potential of darkfield X‐ray tomography is discussed.     

Multimodal hard X‐ray imaging of a mammography phantom at a compact synchrotron light source

The Compact Light Source is a miniature synchrotron producing X‐rays at the interaction point of a counter‐propagating laser pulse and electron bunch through the process of inverse Compton scattering. The small transverse size of the luminous region yields a highly coherent beam with an angular divergence of a few milliradians. The intrinsic monochromaticity and coherence of the produced X‐rays can be exploited in high‐sensitivity differential phase‐contrast imaging with a grating‐based interferometer. Here, the first multimodal X‐ray imaging experiments at the Compact Light Source at a clinically compatible X‐ray energy of 21 keV are reported. Dose‐compatible measurements of a mammography phantom clearly demonstrate an increase in contrast attainable through differential phase and dark‐field imaging over conventional attenuation‐based projections.     

Towards tender X‐rays with Zernike phase‐contrast imaging of biological samples at 50 nm resolution

X‐ray microscopy is a commonly used method especially in material science application, where the large penetration depth of X‐rays is necessary for three‐dimensional structural studies of thick specimens with high‐Z elements. In this paper it is shown that full‐field X‐ray microscopy at 6.2 keV can be utilized for imaging of biological specimens with high resolution. A full‐field Zernike phase‐contrast microscope based on diffractive optics is used to study lipid droplet formation in hepatoma cells. It is shown that the contrast of the images is comparable with that of electron microscopy, and even better contrast at tender X‐ray energies between 2.5 keV and 4 keV is expected.     

Comparative study of multilayers used in monochromators for synchrotron‐based coherent hard X‐ray imaging

A systematic study is presented in which multilayers of different composition (W/Si, Mo/Si, Pd/B₄C), periodicity (from 2.5 to 5.5 nm) and number of layers have been characterized. In particular, the intrinsic quality (roughness and reflectivity) as well as the performance (homogeneity and coherence of the outgoing beam) as a monochromator for synchrotron radiation hard X‐ray micro‐imaging are investigated. The results indicate that the material composition is the dominating factor for the performance. By helping scientists and engineers specify the design parameters of multilayer monochromators, these results can contribute to a better exploitation of the advantages of multilayer monochromators over crystal‐based devices; i.e. larger spectral bandwidth and high photon flux density, which are particularly useful for synchrotron‐based micro‐radiography and ‐tomography.     

A method of hard X‐ray phase‐shifting digital holography

A new method of phase‐shifting digital holography is demonstrated in the hard X‐ray region. An in‐line‐type phase‐shifting holography setup was installed in a 6.80 keV hard X‐ray synchrotron beamline. By placing a phase plate consisting of a hole and a band at the focusing point of a Fresnel lens, the relative phase of the reference and objective beams could be successfully shifted for use with a three‐step phase‐shift algorithm. The system was verified by measuring the shape of a gold test pattern and a silica sphere.     

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.     

Confocal full‐field X‐ray microscope for novel three‐dimensional X‐ray imaging

A confocal full‐field X‐ray microscope has been developed for use as a novel three‐dimensional X‐ray imaging method. The system consists of an X‐ray illuminating `sheet‐beam' whose beam shape is micrified only in one dimension, and an X‐ray full‐field microscope whose optical axis is normal to the illuminating sheet beam. An arbitral cross‐sectional region of the object is irradiated by the sheet‐beam, and secondary X‐ray emission such as fluorescent X‐rays from this region is imaged simultaneously using the full‐field microscope. This system enables a virtual sliced image of a specimen to be obtained as a two‐dimensional magnified image, and three‐dimensional observation is available only by a linear translation of the object along the optical axis of the full‐field microscope. A feasibility test has been carried out at beamline 37XU of SPring‐8. Observation of the three‐dimensional distribution of metallic inclusions in an artificial diamond was performed.     

Alignment of low‐dose X‐ray fluorescence tomography images using differential phase contrast

X‐ray fluorescence nanotomography provides unprecedented sensitivity for studies of trace metal distributions in whole biological cells. Dose fractionation, in which one acquires very low dose individual projections and then obtains high statistics reconstructions as signal from a voxel is brought together (Hegerl & Hoppe, 1976), requires accurate alignment of these individual projections so as to correct for rotation stage runout. It is shown here that differential phase contrast at 10.2 keV beam energy offers the potential for accurate cross‐correlation alignment of successive projections, by demonstrating that successive low dose, 3 ms per pixel, images acquired at the same specimen position and rotation angle have a narrower and smoother cross‐correlation function (1.5 pixels FWHM at 300 nm pixel size) than that obtained from zinc fluorescence images (25 pixels FWHM). The differential phase contrast alignment resolution is thus well below the 700 nm × 500 nm beam spot size used in this demonstration, so that dose fractionation should be possible for reduced‐dose, more rapidly acquired, fluorescence nanotomography experiments.     

Coherent X‐ray diffractive imaging of protein crystals

The technique of coherent X‐ray diffraction imaging (CXDI) has recently shown great promise for the study of inorganic nanocrystals. In this work the CXDI method has been applied to the study of micrometer‐size protein crystals. Finely sampled diffraction patterns of single crystals were measured and iterative phase‐retrieval algorithms were used to reconstruct the two‐dimensional shape of the crystal. The density maps have limited reproducibility because of radiation damage, but show clear evidence for crystal facets. Qualitative analysis of a number of single‐crystal diffraction peaks indicates the presence of inward surface contraction on 2 µm size crystals. A survey of several hundred diffraction patterns yielded a number of examples with dramatic single‐sided streaks, for which a plausible model is constructed.     

Utilizing broadband X‐rays in a Bragg coherent X‐ray diffraction imaging experiment

A method is presented to simplify Bragg coherent X‐ray diffraction imaging studies of complex heterogeneous crystalline materials with a two‐stage screening/imaging process that utilizes polychromatic and monochromatic coherent X‐rays and is compatible with in situ sample environments. Coherent white‐beam diffraction is used to identify an individual crystal particle or grain that displays desired properties within a larger population. A three‐dimensional reciprocal‐space map suitable for diffraction imaging is then measured for the Bragg peak of interest using a monochromatic beam energy scan that requires no sample motion, thus simplifying in situ chamber design. This approach was demonstrated with Au nanoparticles and will enable, for example, individual grains in a polycrystalline material of specific orientation to be selected, then imaged in three dimensions while under load.     

Differential phase contrast with a segmented detector in a scanning X‐ray microprobe

Scanning X‐ray microprobes are unique tools for the nanoscale investigation of specimens from the life, environmental, materials and other fields of sciences. Typically they utilize absorption and fluorescence as contrast mechanisms. Phase contrast is a complementary technique that can provide strong contrast with reduced radiation dose for weakly absorbing structures in the multi‐keV range. In this paper the development of a segmented charge‐integrating silicon detector which provides simultaneous absorption and differential phase contrast is reported. The detector can be used together with a fluorescence detector for the simultaneous acquisition of transmission and fluorescence data. It can be used over a wide range of photon energies, photon rates and exposure times at third‐generation synchrotron radiation sources, and is currently operating at two beamlines at the Advanced Photon Source. Images obtained at around 2 keV and 10 keV demonstrate the superiority of phase contrast over absorption for specimens composed of light elements.     

Image contrast in X‐ray reflection interface microscopy: comparison of data with model calculations and simulations

The contrast mechanism for imaging molecular‐scale features on solid surfaces is described for X‐ray reflection interface microscopy (XRIM) through comparison of experimental images with model calculations and simulated measurements. Images of elementary steps show that image contrast is controlled by changes in the incident angle of the X‐ray beam with respect to the sample surface. Systematic changes in the magnitude and sign of image contrast are asymmetric for angular deviations of the sample from the specular reflection condition. No changes in image contrast are observed when defocusing the condenser or objective lenses. These data are explained with model structure‐factor calculations that reproduce all of the qualitative features observed in the experimental data. These results provide new insights into the image contrast mechanism, including contrast reversal as a function of incident angle, the sensitivity of image contrast to step direction (i.e. up versus down), and the ability to maximize image contrast at almost any scattering condition defined by the vertical momentum transfer, Q_z. The full surface topography can then, in principle, be recovered by a series of images as a function of incident angle at fixed momentum transfer. Inclusion of relevant experimental details shows that the image contrast magnitude is controlled by the intersection of the reciprocal‐space resolution function (i.e. controlled by numerical aperture of the condenser and objective lenses) and the spatially resolved interfacial structure factor of the object being imaged. Together these factors reduce the nominal contrast for a step near the specular reflection condition to a value similar to that observed experimentally. This formalism demonstrates that the XRIM images derive from limited aperture contrast, and explains how non‐zero image contrast can be obtained when imaging a pure phase object corresponding to the interfacial topography.