X-ray microtomography of biological tissues using laboratory and synchrotron sources

X-ray microradiography is a well established technique for the study of biological structures in which the projected absorption is measured, usually with photographic film or resist. If scanning X-ray microradiography with a 15-μm beam, 2-D scanning, and photon counting is used, more accurate results can be obtained and real-time experiments undertaken. Addition of a rotation axis allows computerized axial tomography to be done at a resolution of 15 μm. This technique overcomes the inherent difficulty of microradiography that all detail perpendicular to the plane of the specimen is superimposed. This method has been applied to the study of the 3-D mineral distribution in a 0.8×0.8 mm column of human cortical bone with a laboratory X-ray source. Calculation of the wavelength dependence of the linear absorption coefficient for liver and bone shows that, for a choice of wavelength in the range of 3–0.4 Å (4–30 keV), the specimen thickness can be from 100μm–2 cm and 10 μm–3 mm, respectively. Synchrotron X-radiation has the potential for better resolution because of the higher intensity, which allows the use of a narrower beam. There is also the possibility of determining individual element 3-D distributions from measurements on either side of the absorption edges because of the continuous nature of the spectrum and also the possibility of doing this from X-ray fluorescence measurements. To investigate these possibilities, a tomographic apparatus has been built based on the availability of accurately ground, tungsten carbide balls. Metrological assessment shows that the specimen remains within <1 μm of the required position during translation and rotation. Preliminary X-ray tomographic studies with a 4-μm diameter beam have been started at the Daresbury laboratory synchrotron source.     

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.     

Gold coated contact lens-type ocular in vivo dosimeter (CLOD) for monitoring of low dose in computed tomography: A Monte Carlo study

PurposeThis study reports a sensitivity enhancement of gold-coated contact lens-type ocular in vivo dosimeters (CLODs) for low-dose measurements in computed tomography (CT).MethodsMonte Carlo (MC) simulations were conducted to evaluate the dose enhancement from the gold (Au) layers on the CLODs. The human eye and CLODs were modeled, and the X-ray tube voltages were defined as 80, 120, and 140 kVp. The thickness of the Au layer attached to a CLOD ranged from 100 nm to 10 μm. The thickness of the active layer ranged from 20 to 140 μm. The dose ratio between the active layer of the Au-coated CLOD and a CLOD without a layer, i.e., the dose enhancement factor (DEF), was calculated.ResultsThe DEFs of the first 20-μm thick active layer of the 5-μm thick Au-coated CLOD were 18.4, 19.7, 20.2 at 80, 120, and 140 kVp, respectively. The DEFs decreased as the thickness of the active layer increased. The DEFs of 100-nm to 5-μm thick Au layers increased from 1.7 to 5.4 for 120-kVp X-ray tube voltage when the thickness of the active layer was 140 μm.ConclusionsThe MC results presented a higher sensitivity of Au-coated CLODs (∼20-times higher than that of CLODs without a gold layer). Au-coated CLODs can be applied to an evaluation of very low doses (a few cGy) delivered to patients during CT imaging.     

小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立

目的:利用单碱基编辑技术定点修饰MECP2基因T158M位点,获得单一定点突变类型的啮齿类及非人灵长类胚胎,为建立模拟临床上Rett综合征的疾病动物模型奠定基础。方法:利用单碱基编辑技术构建3对T158M-sgRNA质粒,并分别与BE系统(BE3或BE4max)质粒共转染293T细胞筛选高效的sgRNA,通过显微注射将体外转录的mRNA以不同的浓度组合注射到ICR小鼠和猕猴受精卵中,检测小鼠和猕猴胚胎发育率和编辑效率,评估BE3和BE4max的工作效率及最优浓度组合。结果:小鼠胚胎上BE4max(ng/μl)∶sgRNA(ng/μl)=100∶50浓度组合时,小鼠胚胎发育率和编辑效率最佳,同时该浓度组合在猕猴胚胎上也获得了有效编辑效率。结论:成功在小鼠及猕猴胚胎上实现了MECP2基因T158M位点上C→T的转变,为后期建立T158M突变的RTT动物模型建立了稳定的胚胎编辑体系。     

Investigation of the dose distribution for a cone beam CT system dedicated to breast imaging

Cone-beam breast Computed Tomography (bCT) is an X-ray imaging technique for breast cancer diagnosis, in principle capable of delivering a much more homogeneous dose spatial pattern to the breast volume than conventional mammography, at dose levels comparable to two-view mammography. We present an investigation of the three-dimensional dose distribution for a cone-beam CT system dedicated to breast imaging. We employed Monte Carlo simulations for estimating the dose deposited within a breast phantom having a hemiellipsoidal shape placed on a cylinder of 3.5 cm thickness that simulates the chest wall. This phantom represents a pendulant breast in a bCT exam with the average diameter at chest wall, assumed to correspond to a 5-cm-thick compressed breast in mammography. The phantom is irradiated in a circular orbit with an X-ray cone beam selected from four different techniques: 50, 60, 70, and 80 kVp from a tube with tungsten anode, 1.8 mm Al inherent filtration and additional filtration of 0.2 mm Cu. Using the Monte Carlo code GEANT4 we simulated a system similar to the experimental apparatus available in our lab. Simulations were performed at a constant free-in-air air kerma at the isocenter (1 μGy); the corresponding total number of photon histories per scan was 288 million at 80 kVp. We found that the more energetic beams provide a more uniform dose distribution than at low energy: the 50 kVp beam presents a frequency distribution of absorbed dose values with a coefficient of variation almost double than that for the 80 kVp beam. This is confirmed by the analysis of the relative dose profiles along the radial (i.e. parallel to the “chest wall”) and longitudinal (i.e. from “chest wall” to “nipple”) directions. Maximum radial deviations are on the order of 25% for the 80 kVp beam, whereas for the 50 kVp beam variations around 43% were observed, with the lowest dose values being found along the central longitudinal axis of the phantom.     

Recent advances in X-ray imaging of breast tissue: From two- to three-dimensional imaging

Breast cancer is a globally widespread disease whose detection has already been significantly improved by the introduction of screening programs. Nevertheless, mammography suffers from low soft tissue contrast and the superposition of diagnostically relevant anatomical structures as well as from low values for sensitivity and specificity especially for dense breast tissue. In recent years, two techniques for X-ray breast imaging have been developed that bring advances for the early detection of breast cancer. Grating-based phase-contrast mammography is a new imaging technique that is able to provide three image modalities simultaneously (absorption-contrast, phase-contrast and dark-field signal). Thus, an enhanced detection and delineation of cancerous structures in the phase-contrast image and an improved visualization and characterization of microcalcifications in the dark-field image is possible. Furthermore, latest studies about this approach show that dose-compatible imaging with polychromatic X-ray sources is feasible. In order to additionally overcome the limitations of projection-based imaging, efforts were also made towards the development of breast computed tomography (BCT), which recently led to the first clinical installation of an absorption-based BCT system. Further research combining the benefits of both imaging technologies is currently in progress. This review article summarizes the latest advances in phase-contrast imaging for the female breast (projection-based and three-dimensional view) with special focus on possible clinical implementations in the future.     

Cryo-soft X-ray tomography: a journey into the world of the native-state cell

One of the ultimate aims of imaging in biology is to achieve molecular localisation in the context of the structure of cells in their native state. Here, we review the current state of the art in cryo-soft X-ray tomography (cryo-SXT), which is the only imaging modality that can provide nanoscale 3D information from cryo-preserved, unstained, whole cells thicker than 1 μm. Correlative cryo-fluorescence and cryo-SXT adds functional information to structure, enabling studies of cellular events that cannot be captured using light, electron or X-ray microscopes alone.     

Aluminium and human breast diseases

The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 ± 28 μg/l) compared with control healthy subjects (mean 131 ± 10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate.     

BMIT facility at the Canadian Light Source: Advances in X-ray phase-sensitive imaging

The BioMedical Imaging and Therapy (BMIT) facility [1,2] located at the Canadian Light Source, provides synchrotron-specific imaging and radiation therapy capabilities. There are two separate beamlines used for experiments: the bending magnet (05B1-1) and the insertion device (05ID-2) beamline.The bending magnet beamline provides access to monochromatic beam spanning a spectral range of 15–40 keV, and the beam is 240 mm wide in the POE-2 experimental hutch. Users can also perform experiments with polychromatic (pink) beam.The insertion device beamline was officially opened for general user program in 2015. The source for the ID beamline is a multi-pole, superconducting 4.3 T wiggler. The high field gives a critical energy over 20 keV. The optics hutches prepare a beam that is 220 mm wide in the last experimental hutch SOE-1. The monochromatic spectral range spans 25–150+ keV. Several different X-ray detectors are available for both beamlines, with resolutions ranging from 2 μm to 200 μm.BMIT provides a number of imaging techniques including standard absorption X-ray imaging, K-edge subtraction imaging (KES), in-line phase contrast imaging (also known as propagation based imaging, PBI) and Diffraction Enhanced Imaging/Analyzer Based Imaging (DEI/ABI), all in either projection or CT mode. PBI and DEI/ABI are particularly important tools for BMIT users since these techniques enable visualization of soft tissue and allow for low dose imaging.     

TRIM16 overexpression induces apoptosis through activation of caspase-2 in cancer cells

TRIM16 exhibits tumour suppressor functions by interacting with cytoplasmic vimentin and nuclear E2F1 proteins in neuroblastoma and squamous cell carcinoma cells, reducing cell migration and replication. Reduced TRIM16 expression in a range of human primary malignant tissues correlates with increased malignant potential. TRIM16 also induces apoptosis in breast and lung cancer cells, by unknown mechanisms. Here we show that overexpression of TRIM16 induces apoptosis in human breast cancer (MCF7) and neuroblastoma (BE(2)-C) cells, but not in non-malignant HEK293 cells. TRIM16 increased procaspase-2 protein levels in MCF7 and induced caspase-2 activity in both MCF7 and BE(2)-C cells. We show that TRIM16 and caspase-2 proteins directly interact in both MCF7 and BE(2)-C cells and co-localise in MCF7 cells. Most importantly, the induction of caspase-2 activity is required for TRIM16 to initiate apoptosis. Our data suggest a novel mechanism by which TRIM16 can promote apoptosis by directly modulating caspase-2 activity.     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

Mass spectrometry imaging with high resolution in mass and space

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.     

Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (< 1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy.     

Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

The ability to differentiate benign metaplasia in Barrett’s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 μm and a field of view up to 2.5 cm, allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett’s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett’s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.     

The application of F magnetic resonance ex vivo imaging of three-dimensional cultured breast cancer cells to study the effect of δ-tocopherol

The aim of this study was to determine the role of δ-tocopherol in breast cancer cell growth ex vivo. Human gland mammary adenocarcinoma (MCF-7) and human T-lymphoblastoid (CEM) cells were cultured in the presence of δ-tocopherol at various concentrations (0-750 μM) for 5 days. We have grown 3D ex vivo breast cancer cell cultures in the hollow fiber bioreactor (HFBR). ¹⁹F magnetic resonance imaging (MRI) was used to evaluate oxygen concentration in the cell suspension and thus its viability.     

Detection of single quantum dots in model organisms with sheet illumination microscopy

Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 μm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.     

Imaging performance of phase-contrast breast computed tomography with synchrotron radiation and a CdTe photon-counting detector

PurposeWithin the SYRMA-CT collaboration based at the ELETTRA synchrotron radiation (SR) facility the authors investigated the imaging performance of the phase-contrast computed tomography (CT) system dedicated to monochromatic in vivo 3D imaging of the female breast, for breast cancer diagnosis.MethodsTest objects were imaged at 38 keV using monochromatic SR and a high-resolution CdTe photon-counting detector. Signal and noise performance were evaluated using modulation transfer function (MTF) and noise power spectrum. The analysis was performed on the images obtained with the application of a phase retrieval algorithm as well as on those obtained without phase retrieval. The contrast to noise ratio (CNR) and the capability of detecting test microcalcification clusters and soft masses were investigated.ResultsFor a voxel size of (60 μm)³, images without phase retrieval showed higher spatial resolution (6.7 mm⁻¹ at 10% MTF) than corresponding images with phase retrieval (2.5 mm⁻¹). Phase retrieval produced a reduction of the noise level and an increase of the CNR by more than one order of magnitude, compared to raw phase-contrast images. Microcalcifications with a diameter down to 130 μm could be detected in both types of images.ConclusionsThe investigation on test objects indicates that breast CT with a monochromatic SR source is technically feasible in terms of spatial resolution, image noise and contrast, for in vivo 3D imaging with a dose comparable to that of two-view mammography. Images obtained with the phase retrieval algorithm showed the best performance in the trade-off between spatial resolution and image noise.     

Hard X-ray fluorescence tomography--an emerging tool for structural visualization

Hard X-ray fluorescence microscopy is well-suited to in-situ investigations of trace metal distributions within whole, unstained, biological tissue, with sub-parts-per-million detection achievable in whole cells. The high penetration of X-rays indicates the use of X-ray fluorescence tomography for structural visualization, and recent measurements have realised sub-500-nm tomography on a 10-μm cell. Limitations of present approaches impact the duration of an experiment and imaging fidelity. Developments in X-ray resolution, detector speed, cryogenic environments, and the incorporation of auxiliary signals are being pursued within the synchrotron community. Several complementary approaches to X-ray fluorescence tomography will be routinely available to the biologist in the near future. We discuss these approaches and review applications of biological relevance.     

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.