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.     

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.     

Micro‐CT versus synchrotron radiation phase contrast imaging of human cochlea

High‐resolution images of the cochlea are used to develop atlases to extract anatomical features from low‐resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption‐based micro‐CT to synchrotron radiation phase contrast imaging (SR‐PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR‐PCI and micro‐CT. Images were visually compared and contrast‐to‐noise ratios (CNRs) were computed from n = 27 regions‐of‐interest (enclosing soft tissue) for quantitative comparisons. Three‐dimensional (3D) models of cochlear internal structures were constructed from SR‐PCI images using a semiautomatic segmentation method. SR‐PCI images provided superior visualization of soft tissue microstructures over conventional micro‐CT images. CNR improved from 7.5 ± 2.5 in micro‐CT images to 18.0 ± 4.3 in SR‐PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR‐PCI images are very promising for developing atlas‐based segmentation methods for postoperative evaluation of cochlear implant surgery.     

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.     

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.     

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.     

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.     

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.     

Synchrotron tomographic images from human lung adenocarcinoma: Three‐dimensional reconstruction and histologic correlations

High‐resolution tomographic images using synchrotron X‐rays are expected to provide detailed reflection of microstructures, thereby allowing for the examination of histologic structures without destruction of the specimen. This study aims to evaluate the synchrotron tomographic images of mixed ground‐glass opacity excised on 5‐mm sections in comparison to pathologic examination. The Institutional Review Board of our institute approved this retrospective study, and written informed consent was obtained from each patient whose lung tissue would be used. Obtained lung cancer specimens were brought to the multiple Wiggler 6C beam line at the Pohang Light Source (PLS‐II) in Korea, and phase contrast X‐ray images were obtained in November 2016. The X‐ray emanated from a bending magnet of the electron storage ring with electron energy of 3 GeV, and a typical beam current was 320 mA. Reconstructed tomographic images were compared with images from histologic slides obtained from the same samples. Pulmonary microstructures including terminal bronchioles, alveolar sacs, and vasculature were identified with phase contrast X‐ray images. Images from normal lung tissue and mixed ground‐glass opacity were clearly distinguishable. Hyperplasia of the interalveolar septum and dysplasia of microstructure were clearly identified. The imaging findings correlated well with hematoxylin‐eosin stained specimens. Tomographic images using synchrotron radiation have the potential for clinical applications. With refinement, this technique may become a diagnostic tool for detection of lung cancer.     

Visualization of water drying in porous materials by X‐ray phase contrast imaging

We present in this study results from X‐ray tomographic microscopy with synchrotron radiation performed both in attenuation and phase contrast modes on a limestone sample during two stages of water drying. No contrast agent was used in order to increase the X‐ray attenuation by water. We show that only by using the phase contrast mode it is possible to achieve enough water content change resolution to investigate the drying process at the pore‐scale. We performed 3D image analysis of the time‐differential phase contrast tomogram. We show by the results of such analysis that it is possible to obtain a reliable characterization of the spatial redistribution of water in the resolved pore system in agreement with what expected from the theory of drying in porous media and from measurements performed with other approaches. We thus show the potential of X‐ray phase contrast imaging for pore‐scale investigations of reactive water transport processes which cannot be imaged by adding a contrast agent for exploiting the standard attenuation contrast imaging mode.     

Three‐dimensional visualization of rat retina by X‐ray differential phase contrast tomographic microscopy

The retina is one of the most tiny and sophisticated tissues of the body. Three dimensional (3D) visualization of the whole retina is valuable both in clinical and research arenas. The tissue has been predominantly assessed by time‐consuming histopathology and optical coherence tomography (OCT) in research and clinical arenas. However, none of the two methods can provide 3D imaging of the retina. The purpose of this study is to give a volumetric visualization of rat retina at submicron resolution, using an emerging imaging technique‐phase‐contrast X‐ray CT. A Sprague‐Dawley (SD) rat eye specimen was scanned with X‐ray differential phase contrast tomographic microscopy (DPC‐microCT) equipped at the Swiss Light Source synchrotron. After scanning, the specimen was subjected to routine histology procedures and severed as a reference. The morphological characteristics and signal features of the retina in the DPC‐microCT images were evaluated. The total retina and its sublayers thicknesses were measured on the DPC‐microCT images and compared with those obtained from the histological sections. The retina structures revealed by DPC‐microCT were highly consistent with the histological section. In this study, we achieved nondestructive 3D visualization of SD rat retina. In addition to detailed anatomical structures, the objective parameters provided by DPC‐microCT make it a useful tool for retinal research and disease diagnosis in the early stage.     

Imaging of cochlear tissue with a grating interferometer and hard X‐rays

This article addresses an important current development in medical and biological imaging: the possibility of imaging soft tissue at resolutions in the micron range using hard X‐rays. Challenging environments, including the cochlea, require the imaging of soft tissue structure surrounded by bone. We demonstrate that cochlear soft tissue structures can be imaged with hard X‐ray phase contrast. Furthermore, we show that only a thin slice of the tissue is required to introduce a large phase shift. It is likely that the phase contrast image of the soft tissue structures is sufficient to image the structures even if surrounded by bone. For the present set of experiments, structures with low‐absorption contrast have been visualized using in‐line phase contrast imaging and a grating interferometer. The experiments have been performed at the Advanced Photon Source at Argonne National Laboratories, a third generation source of synchrotron radiation. The source provides highly coherent X‐ray radiation with high‐photon flux (>10¹² photons/s) at high‐photon energies (5–70 keV). Radiographic and light microscopy images of the gerbil cochlear slice samples were compared. It has been determined that a 20‐μm thick tissue slice induces a phase shift between 1/3π and 2/3π. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, 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.     

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.     

Accurate stochastic reconstruction of heterogeneous microstructures by limited x‐ray tomographic projections

An accurate knowledge of the complex microstructure of a heterogeneous material is crucial for its performance prediction, prognosis and optimization. X‐ray tomography has provided a nondestructive means for microstructure characterization in 3D and 4D (i.e. structural evolution over time), in which a material is typically reconstructed from a large number of tomographic projections using filtered‐back‐projection (FBP) method or algebraic reconstruction techniques (ART). Here, we present in detail a stochastic optimization procedure that enables one to accurately reconstruct material microstructure from a small number of absorption contrast x‐ray tomographic projections. This discrete tomography reconstruction procedure is in contrast to the commonly used FBP and ART, which usually requires thousands of projections for accurate microstructure rendition. The utility of our stochastic procedure is first demonstrated by reconstructing a wide class of two‐phase heterogeneous materials including sandstone and hard‐particle packing from simulated limited‐angle projections in both cone‐beam and parallel beam projection geometry. It is then applied to reconstruct tailored Sn‐sphere‐clay‐matrix systems from limited‐angle cone‐beam data obtained via a lab‐scale tomography facility at Arizona State University and parallel‐beam synchrotron data obtained at Advanced Photon Source, Argonne National Laboratory. In addition, we examine the information content of tomography data by successively incorporating larger number of projections and quantifying the accuracy of the reconstructions. We show that only a small number of projections (e.g. 20–40, depending on the complexity of the microstructure of interest and desired resolution) are necessary for accurate material reconstructions via our stochastic procedure, which indicates its high efficiency in using limited structural information. The ramifications of the stochastic reconstruction procedure in 4D materials science are also discussed.     

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.     

Size-selective colloidal-gold localization in transmission X-ray microscopy

Colloidal gold is a useful marker for functional‐imaging experiments in transmission X‐ray microscopy. Due to the low contrast of gold particles with small diameters it is necessary to develop a powerful algorithm to localize the single gold particles. The presented image‐analysis algorithm for identifying colloidal gold particles is based on the combination of a threshold with respect to the local absorption and shape discrimination, realized by fitting a Gaussian profile to the identified regions of interest. The shape discrimination provides the possibility of size‐selective identification and localization of single colloidal gold particles down to a diameter of 50 nm. The image‐analysis algorithm, therefore, has potential for localization studies of several proteins simultaneously and for localization of fiducial markers in X‐ray tomography.     

X-ray omni microscopy

The science of wave‐field phase retrieval and phase measurement is sufficiently mature to permit the routine reconstruction, over a given plane, of the complex wave‐function associated with certain coherent forward‐propagating scalar wave‐fields. This reconstruction gives total knowledge of the information that has been encoded in the complex wave‐field by passage through a sample of interest. Such total knowledge is powerful, because it permits the emulation in software of the subsequent action of an infinite variety of coherent imaging systems. Such ‘virtual optics’, in which software forms a natural extension of the ‘hardware optics’ in an imaging system, may be useful in contexts such as quantitative atom and X‐ray imaging, in which optical elements such as beam‐splitters and lenses can be realized in software rather than optical hardware. Here, we develop the requisite theory to describe such hybrid virtual‐physical imaging systems, which we term ‘omni optics’ because of their infinite flexibility. We then give an experimental demonstration of these ideas by showing that a lensless X‐ray point projection microscope can, when equipped with the appropriate software, emulate an infinite variety of optical imaging systems including those which yield interferograms, Zernike phase contrast, Schlieren imaging and diffraction‐enhanced imaging.     

Exploring the use of soft X‐ray microscopy for imaging subcellular structures of the inner ear1

The soft X‐ray microscope at the Lawrence Berkeley National Laboratory was developed for visualization of biological tissue. Soft X‐ray microscopy provides high‐resolution visualization of hydrated, non‐embedded and non‐sectioned cells and is thus potentially an alternative to transmission electron microscopy. Here we show for the first time soft X‐ray micrographs of structures isolated from the guinea‐pig inner ear. Sensory outer hair cells and supporting pillar cells are readily visualized. In the hair cells, individual stereocilia can easily be identified within the apical hair bundle. The underlying cuticular plate is, however, too densely composed or too thick to be clearly visualized, and thus appears very dark. The cytoplasmic structures protruding from the cuticular plates as well as the fibrillar material surrounding and projecting from the cell nuclei can be seen. In the pillar cells the images reveal individual microtubule bundles. Soft X‐ray images of the acellular tectorial membrane and thin two‐layered Reissner's membrane display a level of resolution comparable to low‐power electron microscopy.     

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.