Imaging Liver Lesions Using Grating-Based Phase-Contrast Computed Tomography with Bi-Lateral Filter Post-Processing

X-ray phase-contrast imaging shows improved soft-tissue contrast compared to standard absorption-based X-ray imaging. Especially the grating-based method seems to be one promising candidate for clinical implementation due to its extendibility to standard laboratory X-ray sources. Therefore the purpose of our study was to evaluate the potential of grating-based phase-contrast computed tomography in combination with a novel bi-lateral denoising method for imaging of focal liver lesions in an ex vivo feasibility study. Our study shows that grating-based phase-contrast CT (PCCT) significantly increases the soft-tissue contrast in the ex vivo liver specimens. Combining the information of both signals – absorption and phase-contrast – the bi-lateral filtering leads to an improvement of lesion detectability and higher contrast-to-noise ratios. The normal and the pathological tissue can be clearly delineated and even internal structures of the pathological tissue can be visualized, being invisible in the absorption-based CT alone. Histopathology confirmed the presence of the corresponding findings in the analyzed tissue. The results give strong evidence for a sufficiently high contrast for different liver lesions using non-contrast-enhanced PCCT. Thus, ex vivo imaging of liver lesions is possible with a polychromatic X-ray source and at a spatial resolution of ∼100 µm. The post-processing with the novel bi-lateral denoising method improves the image quality by combining the information from the absorption and the phase-contrast images.     

Dark-Field Imaging: Recent developments and potential clinical applications

This paper describes an X-ray phase contrast imaging technique using analyzer-based optics called X-ray Dark-Field Imaging that has been under development for the past 10 years. We describe the theory behind XDFI, the X-ray optics required for implementing it in practice, and algorithms used for 2D, 2.5D, and 3D image reconstruction. The XDFI optical chain consists of an asymmetrically cut, Bragg-type monochromator-collimator that provides a planar monochromatic X-ray beam, a positioning stage for the specimens, a Laue-case angle analyzer, and one or two cameras to capture the dark and bright field images. We demonstrate the soft-tissue discrimination capabilities of XDFI by reconstructing images with absorption and phase contrast. By using a variety of specimens such as breast tissue with cancer, joints with articular cartilage, ex-vivo human eye specimen, and others, we show that refraction-based contrast derived from XDFI is more effective in characterizing anatomical features, articular pathology, and neoplastic disease than conventional absorption-based images. For example, XDFI of breast tissue can discriminate between the normal and diseased terminal duct lobular unit, and between invasive and in-situ cancer. The final section of this paper is devoted to potential future developments to enable clinical and histo-pathological applications of this technique.     

Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography

We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from ～6 to 12μm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.     

Structure and flexibility of individual immunoglobulin G molecules in solution

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.     

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of > 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.     

Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging

X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. In this paper, we describe the X-ray optical set-up and the design of the cryo full-field transmission X-ray microscope (TXM) at the electron storage ring BESSY II. Compared to previous TXM set-ups with zone plate condenser monochromator, the new X-ray optical layout employs an undulator source, a spherical grating monochromator and an elliptically shaped glass capillary mirror as condenser. This set-up improves the spectral resolution by an order of magnitude. Furthermore, the partially coherent object illumination improves the contrast transfer of the microscope compared to incoherent conditions. With the new TXM, cells grown on flat support grids can be tilted perpendicular to the optical axis without any geometrical restrictions by the previously required pinhole for the zone plate monochromator close to the sample plane. We also developed an incorporated fluorescence light microscope which permits to record fluorescence, bright field and DIC images of cryogenic cells inside the TXM. For TXM tomography, imaging with multi-keV X-rays is a straightforward approach to increase the depth of focus. Under these conditions phase contrast imaging is necessary. For soft X-rays with shrinking depth of focus towards 10nm spatial resolution, thin optical sections through a thick specimen might be obtained by deconvolution X-ray microscopy. As alternative 3-D X-ray imaging techniques, the confocal cryo-STXM and the dual beam cryo-FIB/STXM with photoelectron detection are proposed.     

Phase-Contrast Hounsfield Units of Fixated and Non-Fixated Soft-Tissue Samples

X-ray phase-contrast imaging is a novel technology that achieves high soft-tissue contrast. Although its clinical impact is still under investigation, the technique may potentially improve clinical diagnostics. In conventional attenuation-based X-ray computed tomography, radiological diagnostics are quantified by Hounsfield units. Corresponding Hounsfield units for phase-contrast imaging have been recently introduced, enabling a setup-independent comparison and standardized interpretation of imaging results. Thus far, the experimental values of few tissue types have been reported; these values have been determined from fixated tissue samples. This study presents phase-contrast Hounsfield units for various types of non-fixated human soft tissues. A large variety of tissue specimens ranging from adipose, muscle and connective tissues to liver, kidney and pancreas tissues were imaged by a grating interferometer with a rotating-anode X-ray tube and a photon-counting detector. Furthermore, we investigated the effects of formalin fixation on the quantitative phase-contrast imaging results.     

Visualizing the internal structure of polyethylene glycol-impregnated wood by digital breast tomosynthesis

This study proposes digital breast tomosynthesis (DBT) as a low-tube-voltage method for imaging wood artifacts treated with polyethylene glycol (PEG). In case of general clinical X-ray Computed Tomography (X-CT), PEG-impregnated wood images typically suffer from low contrast between the PEG and the tree-ring. Because X-CT uses high-tube-voltage X-rays that have high energy, they are transmitted regardless of the X-ray absorption difference of the substance, and therefore, it is not suitable for imaging PEG-impregnated wood. Mammography uses low-tube-voltage X-rays, and therefore, it is suitable for delineating substances with small X-ray absorption differences. However, although mammography can produce high-contrast images of wood, it cannot distinguish three-dimensional (3D) structures such as tree rings, because those are projection images. DBT is a type of mammography used to enhance contrast using low-tube voltage, and it enables imaging 3D structures by exposure X-rays to objects several times changing the exposure angle, and it can obtain quasi-computed tomography. Therefore, we believe that by applying DBT to dendroarchaeology, it would be possible to obtain high-contrast, high-resolution images in the visualization of the internal structure of wood.In this study, we used clinical X-CT, mammography, and DBT to obtain images of wood after PEG impregnation, and we evaluated the internal structure of the wood and the visibility of annual rings. We obtained DBT images as a tomogram with a thickness of 1 mm, which eliminated the distortion of tree rings in the sagittal direction and duplication of the PEG and the tree-ring. Further, tree-rings were easily visualized without a noticeable blur, and the DBT contrast was improved compared to clinical X-CT contrast because DBT was performed at low voltage. Important wooden artifacts excavated from ruins were preserved by PEG. Therefore, this method can be expected to become a very useful tool for dendroarchaeology when used as a complementary tool for microfocus X-CT.     

Mapping 70S ribosomes in intact cells by cryoelectron tomography and pattern recognition

Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7 nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes.     

Zernike phase contrast cryo-electron tomography of whole mounted frozen cells

Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.     

Improved specimen reconstruction by Hilbert phase contrast tomography

The low signal-to-noise ratio (SNR) in images of unstained specimens recorded with conventional defocus phase contrast makes it difficult to interpret 3D volumes obtained by electron tomography (ET). The high defocus applied for conventional tilt series generates some phase contrast but leads to an incomplete transfer of object information. For tomography of biological weak-phase objects, optimal image contrast and subsequently an optimized SNR are essential for the reconstruction of details such as macromolecular assemblies at molecular resolution. The problem of low contrast can be partially solved by applying a Hilbert phase plate positioned in the back focal plane (BFP) of the objective lens while recording images in Gaussian focus. Images recorded with the Hilbert phase plate provide optimized positive phase contrast at low spatial frequencies, and the contrast transfer in principle extends to the information limit of the microscope. The antisymmetric Hilbert phase contrast (HPC) can be numerically converted into isotropic contrast, which is equivalent to the contrast obtained by a Zernike phase plate. Thus, in-focus HPC provides optimal structure factor information without limiting effects of the transfer function. In this article, we present the first electron tomograms of biological specimens reconstructed from Hilbert phase plate image series. We outline the technical implementation of the phase plate and demonstrate that the technique is routinely applicable for tomography. A comparison between conventional defocus tomograms and in-focus HPC volumes shows an enhanced SNR and an improved specimen visibility for in-focus Hilbert tomography.     

Volume interpolation of CT images from tree trunks

Computerized tomography as a non-destructive scanning method to analyze wood structures has become an important technique in tree research. The possibility to reconstruct three-dimensional volumes based on a number of slices of two-dimensional data from CT scans is strongly dependent on the number of measured slices. Radial basis function methods can be successfully used to interpolate CT images with the aim of obtaining a satisfactory reconstruction of tree trunks. In contrast to standard interpolation techniques, our method takes into account that wood structures differ more in the radial than in the longitudinal direction. Therefore we obtain better interpolation results for wood structures.     

Computer tomography of the brain in Hamilton.

Computer tomography, a new noninvasive, rapid and easily tolerated technique of brain examination, has been evaluated by analysis of 1000 examinations. It is much more sensitive than conventional radiographic techniques and can resolve soft-tissue structures that differ only slightly in density. It also provides direct visualization of the ventricular system. The range of clinical applications is wide; it is especially useful in differentiating intracerebral hemorrhage from infarction, and in demonstrating many brain tumours, particularly supratentorial, though enhancement with a water-soluble contrast medium injected intravenously is often necessary.     

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

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

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

Non-invasive assessment of soft-tissue artifact and its effect on knee joint kinematics during functional activity

The soft-tissue interface between skin-mounted markers and the underlying bones poses a major limitation to accurate, non-invasive measurement of joint kinematics. The aim of this study was twofold: first, to quantify lower limb soft-tissue artifact in young healthy subjects during functional activity; and second, to determine the effect of soft-tissue artifact on the calculation of knee joint kinematics. Subject-specific bone models generated from magnetic resonance imaging (MRI) were used in conjunction with X-ray images obtained from single-plane fluoroscopy to determine three-dimensional knee joint kinematics for four separate tasks: open-chain knee flexion, hip axial rotation, level walking, and a step-up. Knee joint kinematics was derived using the anatomical frames from the MRI-based, 3D bone models together with the data from video motion capture and X-ray fluoroscopy. Soft-tissue artifact was defined as the degree of movement of each marker in the anteroposterior, proximodistal and mediolateral directions of the corresponding anatomical frame. A number of different skin-marker clusters (total of 180) were used to calculate knee joint rotations, and the results were compared against those obtained from fluoroscopy. Although a consistent pattern of soft-tissue artifact was found for each task across all subjects, the magnitudes of soft-tissue artifact were subject-, task- and location-dependent. Soft-tissue artifact for the thigh markers was substantially greater than that for the shank markers. Markers positioned in the vicinity of the knee joint showed considerable movement, with root mean square errors as high as 29.3 mm. The maximum root mean square errors for calculating knee joint rotations occurred for the open-chain knee flexion task and were 24.3°, 17.8° and 14.5° for flexion, internal–external rotation and abduction–adduction, respectively. The present results on soft-tissue artifact, based on fluoroscopic measurements in healthy adult subjects, may be helpful in developing location- and direction-specific weighting factors for use in global optimization algorithms aimed at minimizing the effects of soft-tissue artifact on calculations of knee joint rotations.     

EGS_cbct: Simulation of a fan beam CT and RMI phantom for measured HU verification

IntroductionA mathematical 3D model of an existing computed tomography (CT) scanner was created and used in the EGSnrc-based BEAMnrc and egs_cbct Monte Carlo codes. Simulated transmission dose profiles of a RMI-465 phantom were analysed to verify Hounsfield numbers against measured data obtained from the CT scanner.Methods and materialsThe modelled CT unit is based on the design of a Toshiba Aquilion 16 LB CT scanner. As a first step, BEAMnrc simulated the X-ray tube, filters, and secondary collimation to obtain phase space data of the X-ray beam. A bowtie filter was included to create a more uniform beam intensity and to remove the beam hardening effects. In a second step the Interactive Data Language (IDL) code was used to build an EGSPHANT file that contained the RMI phantom which was used in egs_cbct simulations. After simulation a series of profiles were sampled from the detector model and the Feldkamp-Davis-Kress (FDK) algorithm was used to reconstruct transversal images. The results were tested against measured data obtained from CT scans.ResultsThe egs_cbct code can be used for the simulation of a fan beam CT unit. The calculated bowtie filter ensured a uniform flux on the detectors. Good correlation between measured and simulated CT numbers was obtained.ConclusionsIn principle, Monte Carlo codes such as egs_cbct can model a fan beam CT unit. After reconstruction, the images contained Hounsfield values comparable to measured data.     

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

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

Tomographic techniques for the study of exceptionally preserved fossils

Three-dimensional fossils, especially those preserving soft-part anatomy, are a rich source of palaeontological information; they can, however, be difficult to work with. Imaging of serial planes through an object (tomography) allows study of both the inside and outside of three-dimensional fossils. Tomography may be performed using physical grinding or sawing coupled with photography, through optical techniques of serial focusing, or using a variety of scanning technologies such as neutron tomography, magnetic resonance imaging and most usefully X-ray computed tomography. This latter technique is applicable at a variety of scales, and when combined with a synchrotron X-ray source can produce very high-quality data that may be augmented by phase-contrast information to enhance contrast. Tomographic data can be visualized in several ways, the most effective of which is the production of isosurface-based 'virtual fossils' that can be manipulated and dissected interactively.