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 single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

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.     

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.     

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.     

Soft X‐ray spectromicroscopy for speciation,quantitation and nano‐eco‐toxicology of nanomaterials

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X‐ray absorption properties, soft X‐ray scanning transmission X‐ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal‐containing nanomaterials (Ti, Ni, Cu) and carbon‐based nanomaterials (multiwalled carbon nanotubes, C₆₀ fullerene). The benefits of X‐ray fluorescence detection in soft X‐ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.     

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.     

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.     

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

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.     

Towards the imaging of Weibel–Palade body biogenesis by serial block face‐scanning electron microscopy

Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face‐scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three‐dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre‐scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three‐dimensional analysis. Here, we explore focused ion beam facilitated serial block face‐scanning electron microscopy to study the endothelial cell–specific storage organelles, the Weibel–Palade bodies, during their biogenesis at the Golgi apparatus. Weibel–Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face‐scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel–Palade bodies and Golgi stacks which became conceivable in large‐scale three‐dimensional data. We demonstrate that serial block face‐scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.     

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.     

Investigation of inner ear anatomy in mouse using X‐ray phase contrast tomography

A thorough understanding of inner ear anatomy is important for investigators. However, investigation of the mouse inner ear is difficult due to the limitations of imaging techniques. X‐ray phase contrast tomography increases contrast 100–1,000 times compared with conventional X‐ray imaging. This study aimed to investigate inner ear anatomy in a fresh post‐mortem mouse using X‐ray phase contrast tomography and to provide a comprehensive atlas of microstructures with less tissue deformation. All experiments were performed in accordance with our institution's guidelines on the care and use of laboratory animals. A fresh mouse cadaver was scanned immediately after sacrifice using an inline phase contrast tomography system. Slice images were reconstructed using a filtered back‐projection (FBP) algorithm. Standardized axial and coronal planes were adjusted with a multi‐planar reconstruction method. Some three‐dimensional (3D) objects were reconstructed by surface rendering. The characteristic features of microstructures, including otoconia masses of the saccular and utricular maculae, superior and inferior macula cribrosae, single canal, modiolus, and osseous spiral lamina, were described in detail. Spatial positions and relationships of the vestibular structures were exhibited in 3D views. This study investigated mouse inner ear anatomy and provided a standardized presentation of microstructures. In particular, otoconia masses were visualized in their natural status without contrast for the first time. The comprehensive anatomy atlas presented in this study provides an excellent reference for morphology studies of the inner ear.     

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.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

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 nanoparticle‐algae interactions in three dimensions using Cytoviva microscopy

Plasmonic resonances of metal‐based nanoparticles are increasingly used for ultrasensitive imaging assays. In this context, the Cytoviva^TM microscopy platform has greatly gained in popularity. In essence, Cytoviva is an optimized dark field microscope that permits detection of particles down to a few nanometers in size. A significant limitation of Cytoviva up to now has been that it only provided for single plane imaging. The datasets produced by this technique therefore only show a partial view of the sample – not ideally suited to analysis. Here we explain how to overcome this limitation by mounting the Cytoviva condenser on an automated microscope with Z‐scanning capability. Our method allows three‐dimensional mapping of nanoparticles in their full three‐dimensional cellular context. We apply this technique to study the interaction of silver and cerium dioxide nanoparticles with cells of the green alga, Pseudokirchneriella subcapitata, a system of significant environmental relevance because algae underlie much of the aquatic food chain. Our objective was to develop a technique to visualize in detail the interaction of nanoparticles with cells in three dimensions, such that one may, for example, determine whether a particular nanoparticle is inside a cell, at its very surface, or at a distance from it.     

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.     

Cyclic fatigue resistance and energy dispersive X‐ray spectroscopy analysis of novel heat‐treated nickel–titanium instruments at body temperature

The aim of this study was to evaluate the cyclic fatigue resistance of Dia‐X, WaveOne Gold and One Curve files in a water bath at intracanal temperature (35°C). Thirty‐nine instruments Dia‐X, WaveOne Gold, and One Curve systems (n = 13) were tested in an artificial canal with a curvature angle of 60° and a radius of 3 mm. A water bath setup at a temperature of 35°C was used to simulate the intracanal temperature and time to fracture (TTF) as seconds was recorded. The mean data were analyzed statistically using one‐way ANOVA, and post hoc Tukey test (p = .05). The fractured surface of the instruments was examined with scanning electron microscopy (SEM) and chemical composition of the instruments were investigated with energy dispersive X‐ray spectroscopy (EDS). Statistically significant differences were detected in TTF values of all the systems as follows: One Curve > WaveOne Gold > Dia‐X (p < .05). One Curve instruments demonstrated the highest TTF values in all the tested instruments. The EDS microanalysis revealed similar NiTi composition of on the surface of One Curve, WaveOne Gold, and Dia‐X instruments. The novel manufacturing process, including C‐wire heat treatment and the variable cross‐section of the One Curve files, could be the main factors affecting the fatigue life of the instruments.