Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

Imaging connected porosity of crystalline rock by contrast agent‐aided X‐ray microtomography and scanning electron microscopy

We set out to study connected porosity of crystalline rock using X‐ray microtomography and scanning electron microscopy with energy dispersive X‐ray spectroscopy (SEM‐EDS) with caesium chloride as a contrast agent. Caesium is an important radionuclide regarding the final deposition of nuclear waste and also forms dense phases that can be readily distinguished by X‐ray microtomography and SEM‐EDS. Six samples from two sites, Olkiluoto (Finland) and Grimsel (Switzerland), where transport properties of crystalline rock are being studied in situ, were investigated using X‐ray microtomography and SEM‐EDS. The samples were imaged with X‐ray microtomography, immersed in a saturated caesium chloride (CsCl) solution for 141, 249 and 365 days and imaged again with X‐ray microtomography. CsCl inside the samples was successfully detected with X‐ray microtomography and it had completely penetrated all six samples. SEM‐EDS elemental mapping was used to study the location of caesium in the samples in detail with quantitative mineral information. Precipitated CsCl was found in the connected pore space in Olkiluoto veined gneiss and in lesser amounts in Grimsel granodiorite. Only a very small amount of precipitated CsCl was observed in the Grimsel granodiorite samples. In Olkiluoto veined gneiss caesium was found in pinitised areas of cordierite grains. In the pinitised areas caesium was found in notable excess compared to chloride, possibly due to the combination of small pore size and negatively charged surfaces. In addition, elevated concentrations of caesium were found in kaolinite and sphalerite phases. The findings concerning the location of CsCl were congruent with X‐ray microtomography.     

Registration of phase‐contrast images in propagation‐based X‐ray phase tomography

X‐ray phase tomography aims at reconstructing the 3D electron density distribution of an object. It offers enhanced sensitivity compared to attenuation‐based X‐ray absorption tomography. In propagation‐based methods, phase contrast is achieved by letting the beam propagate after interaction with the object. The phase shift is then retrieved at each projection angle, and subsequently used in tomographic reconstruction to obtain the refractive index decrement distribution, which is proportional to the electron density. Accurate phase retrieval is achieved by combining images at different propagation distances. For reconstructions of good quality, the phase‐contrast images recorded at different distances need to be accurately aligned. In this work, we characterise the artefacts related to misalignment of the phase‐contrast images, and investigate the use of different registration algorithms for aligning in‐line phase‐contrast images. The characterisation of artefacts is done by a simulation study and comparison with experimental data. Loss in resolution due to vibrations is found to be comparable to attenuation‐based computed tomography. Further, it is shown that registration of phase‐contrast images is nontrivial due to the difference in contrast between the different images, and the often periodical artefacts present in the phase‐contrast images if multilayer X‐ray optics are used. To address this, we compared two registration algorithms for aligning phase‐contrast images acquired by magnified X‐ray nanotomography: one based on cross‐correlation and one based on mutual information. We found that the mutual information‐based registration algorithm was more robust than a correlation‐based method.     

Evaluation of X‐ray tomography contrast agents: A review of production,protocols, and biological applications

X‐ray computed tomography is a strong tool that finds many applications both in medical applications and in the investigation of biological and nonbiological samples. In the clinics, X‐ray tomography is widely used for diagnostic purposes whose three‐dimensional imaging in high resolution helps physicians to obtain detailed image of investigated regions. Researchers in biological sciences and engineering use X‐ray tomography because it is a nondestructive method to assess the structure of their samples. In both medical and biological applications, visualization of soft tissues and structures requires special treatment, in which special contrast agents are used. In this detailed report, molecule‐based and nanoparticle‐based contrast agents used in biological applications to enhance the image quality were compiled and reported. Special contrast agent applications and protocols to enhance the contrast for the biological applications and works to develop nanoparticle contrast agents to enhance the contrast for targeted drug delivery and general imaging applications were also assessed and listed.     

Phase‐contrast hard X‐ray microscopy using synchrotron radiation for the properties of skeletal muscle in mouse hind limbs

Radiation beam interface contrast X‐ray microscopy provides resolution of a few dozen nanometers from fixed whole muscle biopsies, allowing better reconstruction of the microstructure of the muscle than is currently possible with classic histological techniques. Fixed soleus muscle biopsies have been evaluated from the walk‐in mouse model using phase‐contrast X‐ray microscopy, and results presented that corroborate the accuracy of the method used, and its potential for application in physiotherapy and occupational therapy studies. We believe that this method will enhance existing morphometric methods of analysis, leading to accurate reconstruction of other thick specimens that would otherwise require thin sectioning and reconstruction through deconvolution algorithms.     

X-ray high-resolution vascular network imaging

This paper presents the first application of high‐resolution X‐ray synchrotron tomography to the imaging of large microvascular networks in biological tissue samples. This technique offers the opportunity of analysing the full three‐dimensional vascular network from the micrometre to the millimetre scale. This paper presents the specific sample preparation method and the X‐ray imaging procedure. Either barium or iron was injected as contrast agent in the vascular network. The impact of the composition and concentration of the injected solution on the X‐ray synchrotron tomography images has been studied. Two imaging modes, attenuation and phase contrast, are compared. Synchrotron high‐resolution computed tomography offers new prospects in the three‐dimensional imaging of in situ biological vascular networks.     

Contact X‐ray microscopy of living cells by using LiF crystal as imaging detector

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single‐shot soft X‐ray contact microscopy is presented. High resolved X‐ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X‐ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid‐state X‐ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X‐ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X‐ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high‐quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.     

Comparison of three‐dimensional analysis and stereological techniques for quantifying lithium‐ion battery electrode microstructures

Lithium‐ion battery performance is intrinsically linked to electrode microstructure. Quantitative measurement of key structural parameters of lithium‐ion battery electrode microstructures will enable optimization as well as motivate systematic numerical studies for the improvement of battery performance. With the rapid development of 3‐D imaging techniques, quantitative assessment of 3‐D microstructures from 2‐D image sections by stereological methods appears outmoded; however, in spite of the proliferation of tomographic imaging techniques, it remains significantly easier to obtain two‐dimensional (2‐D) data sets. In this study, stereological prediction and three‐dimensional (3‐D) analysis techniques for quantitative assessment of key geometric parameters for characterizing battery electrode microstructures are examined and compared. Lithium‐ion battery electrodes were imaged using synchrotron‐based X‐ray tomographic microscopy. For each electrode sample investigated, stereological analysis was performed on reconstructed 2‐D image sections generated from tomographic imaging, whereas direct 3‐D analysis was performed on reconstructed image volumes. The analysis showed that geometric parameter estimation using 2‐D image sections is bound to be associated with ambiguity and that volume‐based 3‐D characterization of nonconvex, irregular and interconnected particles can be used to more accurately quantify spatially‐dependent parameters, such as tortuosity and pore‐phase connectivity.     

Laser‐preparation of geometrically optimised samples for X‐ray nano‐CT

A robust and versatile sample preparation technique for the fabrication of cylindrical pillars for imaging by X‐ray nano‐computed tomography (nano‐CT) is presented. The procedure employs simple, cost‐effective laser micro‐machining coupled with focused‐ion beam (FIB) milling, when required, to yield mechanically robust samples at the micrometre length‐scale to match the field‐of‐view (FOV) for nano‐CT imaging. A variety of energy and geological materials are exhibited as case studies, demonstrating the procedure can be applied to a variety of materials to provide geometrically optimised samples whose size and shape are tailored to the attenuation coefficients of the constituent phases. The procedure can be implemented for the bespoke preparation of pillars for both lab‐ and synchrotron‐based X‐ray nano‐CT investigations of a wide range of samples.     

Synchrotron nanoscopy imaging study of scalp hair in breast cancer patients and healthy individuals: Difference in medulla loss and cortical membrane enhancements

Nanoscopic synchrotron X‐ray imaging was performed on scalp hair samples of patients with breast cancer and healthy individuals to investigate any structural differences as diagnostic tool. Hair strands were divided into 2‐3 segments along the strands from root to tip, followed by imaging either in projection or in CT scanning with a monochromatic 6.78‐keV X‐ray using zone‐plate optics with a resolving power of 60 nm. All the examined cancer hairs exhibited medulla loss with cancer stage‐dependent pattern; complete loss, discontinuous or trace along the strands. In contrast, medullas were well retained without complete loss in the healthy hair. In the CT‐scanned axial images, the cortical spindle compartments had no contrast in the healthy hair, but appeared hypointense in contrast to the surrounding hyperintense cortical membrane complex in the cancer hair. In conclusion, observation of medulla loss and cortical membrane enhancements in the hair strands of breast cancer patients demonstrated structural variations in the cancer hair, providing a new platform for further synchrotron X‐ray imaging study of screening breast cancer patients. Microsc. Res. Tech. 79:23–30, 2016. © 2015 Wiley Periodicals, Inc.     

Quantitative imaging of Candida utilis and its organelles by soft X‐ray Nano‐CT

Soft X‐ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X‐ray microscopy and three‐dimensional volumes were reconstructed with the SART‐TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X‐ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three‐dimensional datasets. With such excellent features, soft X‐ray imaging has a great influence in the field of biological cellular and subcellular research.     

Cryo‐scanning x‐ray diffraction microscopy of frozen‐hydrated yeast

We developed cryo‐scanning x‐ray diffraction microscopy, utilizing hard x‐ray ptychography at cryogenic temperature, for the noninvasive, high‐resolution imaging of wet, extended biological samples and report its first frozen‐hydrated imaging. Utilizing phase contrast at hard x‐rays, cryo‐scanning x‐ray diffraction microscopy provides the penetration power suitable for thick samples while retaining sensitivity to minute density changes within unstained samples. It is dose‐efficient and further minimizes radiation damage by keeping the wet samples at cryogenic temperature. We demonstrate these capabilities in two dimensions by imaging unstained frozen‐hydrated budding yeast cells, achieving a spatial resolution of 85 nm with a phase sensitivity of 0.0053 radians. The current work presents the feasibility of cryo‐scanning x‐ray diffraction microscopy for quantitative, high‐resolution imaging of unmodified biological samples extending to tens of micrometres.     

Imaging biofilm in porous media using X-ray computed microtomography

In this study, a new technique for three-dimensional imaging of biofilm within porous media using X-ray computed microtomography is presented. Due to the similarity in X-ray absorption coefficients for the porous media (plastic), biofilm and aqueous phase, an X-ray contrast agent is required to image biofilm within the experimental matrix using X-ray computed tomography. The presented technique utilizes a medical suspension of barium sulphate to differentiate between the aqueous phase and the biofilm. Potassium iodide is added to the suspension to aid in delineation between the biofilm and the experimental porous medium. The iodide readily diffuses into the biofilm while the barium sulphate suspension remains in the aqueous phase. This allows for effective differentiation of the three phases within the experimental systems utilized in this study. The behaviour of the two contrast agents, in particular of the barium sulphate, is addressed by comparing two-dimensional images of biofilm within a pore network obtained by (1) optical visualization and (2) X-ray absorption radiography. We show that the contrast mixture provides contrast between the biofilm, the aqueous-phase and the solid-phase (beads). The imaging method is then applied to two three-dimensional packed-bead columns within which biofilm was grown. Examples of reconstructed images are provided to illustrate the effectiveness of the method. Limitations and applications of the technique are discussed. A key benefit, associated with the presented method, is that it captures a substantial amount of information regarding the topology of the pore-scale transport processes. For example, the quantification of changes in porous media effective parameters, such as dispersion or permeability, induced by biofilm growth, is possible using specific upscaling techniques and numerical analysis. We emphasize that the results presented here serve as a first test of this novel approach; issues with accurate segmentation of the images, optimal concentrations of contrast agents and the potential need for use of synchrotron radiation sources need to be addressed before the method can be used for precise quantitative analysis of biofilm geometry in porous media.     

Nanoscale elemental sensitivity study of Nd2Fe14B using absorption correlation tomography

Transmission X‐ray microscopy (TXM) is a rapidly developing technique with the capability of nanoscale three dimensional (3D) real‐space imaging. Combined with the wide range in energy tunability from synchrotron sources, TXM enables the retrieval of 3D microstructural information with elemental/chemical sensitivity that would otherwise be inaccessible. The differential absorption contrast above and below absorption edges has been used to reconstruct the distributions of different elements, assuming the absorption edges of the interested elements are fairly well separated. Here we present an “Absorption Correlation Tomography” (ACT) method based on the correlation of the material absorption across multiple edges. ACT overcomes the significant limitation caused by overlapping absorption edges, significantly expands the capabilities of TXM, and makes it possible for fully quantitative nano‐scale 3D structural investigation with chemical/elemental sensitivity. The capability and robustness of this new methodology is demonstrated in a case study of an important type of rare earth magnet (Nd₂Fe₁₄B). Microsc. Res. Tech. 76:1112–1117, 2013. © 2013 Wiley Periodicals, Inc.     

Evaluation of contrasting techniques for X‐ray imaging of velvet worms (Onychophora)

Non‐invasive imaging techniques like X‐ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft‐bodied invertebrates, remains understudied. Herein, we used synchrotron radiation‐based X‐ray micro‐computed tomography to compare the effects of commonly used contrasting approaches on onychophorans – soft‐bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation‐based and differential phase‐contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal‐to‐noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box‐and‐whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X‐ray micro‐computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora.     

An exploratory study of contrast agents for soft tissue visualization by means of high resolution X‐ray computed tomography imaging

High resolution X‐ray computed tomography (CT), or microCT, is a promising and already widely used technique in various scientific fields. Also for histological purposes it has great potential. Although microCT has proven to be a valuable technique for the imaging of bone structures, the visualization of soft tissue structures is still an important challenge due to their low inherent X‐ray contrast. One way to achieve contrast enhancement is to make use of contrast agents. However, contrary to light and electron microscopy, knowledge about contrast agents and staining procedures is limited for X‐ray CT. The purpose of this paper is to identify useful X‐ray contrast agents for soft tissue visualization, which can be applied in a simple way and are also suited for samples larger than (1 cm)³. And 28 chemical substances have been investigated. All chemicals were applied in the form of concentrated aqueous solutions in which the samples were immersed. First, strips of green Bacon were stained to evaluate contrast enhancement between muscle and adipose tissue. Furthermore it was also tested whether the contrast agents remained fixed in the tissue after staining by re‐immersing them in water. Based on the results, 12 contrast agents were selected for further testing on postmortem mice hind legs, containing a variety of different tissues, including muscle, fat, bone, cartilage and tendons. It was evaluated whether the contrast agents allowed a clearer distinction between the different soft tissue structures present. Finally also penetration depth was measured. And 26 chemicals resulted in contrast enhancement between muscle and adipose tissue in the Bacon strips. Mercury(II)chloride (HgCl₂), phosphotungstic acid (PTA), phosphomolybdic acid (PMA) and ammonium orthomolybdate ((NH₄)₂MoO₄) remained fixed after re‐immersion in water. The penetration tests showed that potassium iodide (KI) and sodium tungstate can be most efficiently used for large samples of the order of several tens of cm³. PMA, PTA, HgCl₂ and also to a lesser extent Na₂WO₄ and (NH₄)₂MoO₄ allowed a clearer distinction between the different soft tissue structures present.     

Structural investigations on green culms and charcoal of Bambusa multiplex

Green culms of Bambusa multiplex and the bamboo charcoal carbonized from the green culms at 700°C have been studied by means of X‐ray diffraction, X‐ray fluorescent element analysis, analytical scanning electron microscopy, and analytical scanning transmission electron microscopy (STEM), aiming at industrial applications as raw materials for functional devices and substances. It is revealed that the green culms and the charcoal contain a significant amount of Si, in particular, ～18 wt % in the skin. The green culms comprise amorphous and crystalline celluloses. The charcoal has a so‐called amorphous structure which is composed of randomly distributed carbon nanotubes and fibers. The growth of Ag‐doped activated charcoal powders that were produced by two different methods using this charcoal powder has also been studied.     

Application of X‐ray microcomputed tomography in the characterization of irradiated nuclear fuel and material specimens

X‐ray microcomputed tomography (μCT) was applied in characterizing the internal structures of a number of irradiated materials, including carbon‐carbon fibre composites, nuclear‐grade graphite and tristructural isotropic‐coated fuel particles. Local cracks in carbon‐carbon fibre composites associated with their synthesis process were observed with μCT without any destructive sample preparation. Pore analysis of graphite samples was performed quantitatively, and qualitative analysis of pore distribution was accomplished. It was also shown that high‐resolution μCT can be used to probe internal layer defects of tristructural isotropic‐coated fuel particles to elucidate the resulting high release of radioisotopes. Layer defects of sizes ranging from 1 to 5 μm and up could be isolated by tomography. As an added advantage, μCT could also be used to identify regions with high densities of radioisotopes to determine the proper plane and orientation of particle mounting for further analytical characterization, such as materialographic sectioning followed by optical and electron microscopy. In fully ceramic matrix fuel forms, despite the highly absorbing matrix, characterization of tristructural isotropic‐coated particles embedded in a silicon carbide matrix was accomplished using μCT and related advanced image analysis techniques.