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.     

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.     

Detection of lead in Zea mays by dual‐energy X‐ray microtomography at the SYRMEP beamline of the ELETTRA synchrotron and by atomic absorption spectroscopy

This study is related to the application of the X‐ray dual‐energy microradiography technique together with the atomic absorption spectroscopy (AAS) for the detection of lead on Zea mays stem, ear, root, and leaf samples. To highlight the places with lead intake, the planar radiographs taken with monochromatic X‐ray radiation in absorption regime with photon energy below and above the absorption edge of a given chemical element, respectively, are analyzed and processed. To recognize the biological structures involved in the intake, the dual‐energy images with the lead signal have been compared with the optical images of the same Z. mays stem. The ear, stem, root, and leaf samples have also been analyzed with the AAS technique to measure the exact amount of the hyperaccumulated lead. The AAS measurement revealed that the highest intake occurred in the roots while the lowest in the maize ears and in the leaf. It seems there is a particular mechanism that protects the seeds and the leaves in the intake process. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc. This article was published online on 1 December 2009. An error was subsequently identified. This notice is included in the online and print version to indicate that both have been corrected 19 February 2010.     

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.     

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.     

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

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

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.     

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.     

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.     

Mistakes encountered during automatic peak identification of minor and trace constituents in electron‐excited energy dispersive X‐ray microanalysis

Automated peak identification in electron beam‐excited X‐ray microanalysis with energy dispersive X‐ray spectrometry has been shown to be subject to occasional mistakes even on well‐separated, high‐intensity peaks arising from major constituents (arbitrarily defined as a concentration, C, which exceeds a mass fraction of 0.1). The peak identification problem becomes even more problematic for constituents present at minor (0.01≤C≤0.1) and trace (C<0.01) levels. “Problem elements” subject to misidentification as major constituents are even more vulnerable to misidentification when present at low concentrations in the minor and trace ranges. Additional misidentifications attributed to trace elements include minor X‐ray family members associated with major constituents but not assigned properly, escape and coincidence peaks associated with major constituents, and false peaks owing to chance groupings of counts in spectra with poor counting statistics. A strategy for robust identification of minor and trace elements can be based on application of automatic peak identification with careful inspection of the results followed by multiple linear least‐squares peak fitting with complete peak references to systematically remove each identified major element from the spectrum before attempting to assign remaining peaks to minor and trace constituents. SCANNING 31: 91–101, 2009. Published by Wiley Periodicals, Inc.     

Synchrotron radiation X‐ray micro‐fluorescence: Protocol to study mesenchymal stem cells

The micro‐X‐ray fluorescence by synchrotron radiation (μ‐XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ‐XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10⁴ to 1 × 10⁷ were divided in two groups: G1: fixed in 4% paraformaldehyde diluted in phosphate‐buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10⁷ cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ‐XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10⁷ cells. Microsc. Res. Tech. 79:149–154, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Cells on biomaterials – some aspects of elemental analysis by means of electron probes

Electron probe X‐ray microanalysis enables concomitant observation of specimens and analysis of their elemental composition. The method is attractive for engineers developing tissue‐compatible biomaterials. Either changes in element composition of cells or biomaterial can be defined according to well‐established preparation and quantification procedures. However, the qualitative and quantitative elemental analysis appears more complicated when cells or thin tissue sections are deposited on biomaterials. X‐ray spectra generated at the cell/tissue–biomaterial interface are modelled using a Monte Carlo simulation of a cell deposited on borosilicate glass. Enhanced electron backscattering from borosilicate glass was noted until the thickness of the biological layer deposited on the substrate reached 1.25 μm. It resulted in significant increase in X‐ray intensities typical for the elements present in the cellular part. In this case, the mean atomic number value of the biomaterial determines the strength of this effect. When elements are present in the cells only, the positive linear relationship appears between X‐ray intensities and cell thickness. Then, spatial dimensions of X‐ray emission for the particular elements are exclusively in the range of the biological part and the intensities of X‐rays become constant. When the elements are present in both the cell and the biomaterial, X‐ray intensities are registered for the biological part and the substrate simultaneously leading to a negative linear relationship of X‐ray intensities in the function of cell thickness. In the case of the analysis of an element typical for the biomaterial, strong decrease in X‐ray emission is observed in the function of cell thickness as the effect of X‐ray absorption and the limited excitation range to biological part rather than to the substrate. Correction procedures for calculations of element concentrations in thin films and coatings deposited on substrates are well established in materials science, but little is known about factors that have to be taken into account to accurately quantify bioelements in thin and semi‐thick biological samples. Thus thorough tests of currently available quantification procedures are required to verify their applicability to cells or tissues deposited on the biomaterials.     

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.     

A graphic approach to the analysis of a large X‐ray microanalysis dataset obtained via SEM–EDS

A graphical method for phase analysis of advanced materials by EDS–SEM was developed and demonstrated on deformed superconducting Bi(Pb)2223 ceramics. Through visual representation, this method allows for the rapid and efficient analysis of large X‐ray microanalysis datasets and to identify phase composition of fine particles of secondary phases against a background of other phases. The graphical method can be applied using existing software and therefore does not require the development of new programs or complex computations.     

X‐ray absorption near‐edge structure analysis of tribofilms generated by highly efficient,friction‐reducing additive in water–polyglycol–glycol lubricant

The development of environment‐friendly lubricant additives can make a valuable contribution to addressing human health and energy problems. In the present work, the compound ammonium tetrathiomolybdate was synthesised and used as a highly efficient, friction‐reducing additive in water–polyglycol–glycol lubricants. Its tribological properties were tested by a four‐ball machine, and the composition of the tribofilm was identified by X‐ray absorption near‐edge structure (XANES) spectroscopy. The tribological test results indicated that the additive cannot only reduce the friction coefficient value, but can also shorten the rubbing time needed to produce a ‘stable friction coefficient value’. The XANES results showed that the tribofilm is mainly composed of an adsorbed layer and a tribochemical layer; the formation of sulphate and molybdenum trioxide in the tribofilm on the metal wear surface is responsible for the excellent anti‐friction performances of the additive. Copyright © 2009 John Wiley & Sons, Ltd.     

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

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

Microporosity and polymerization contraction as function of depth in dental resin cements by X‐ray computed microtomography

This research aimed to obtain the depth dependence of polymerization contraction and microporosity from irradiated dental resin cements by X‐ray computed microtomography (μCT). Samples (n = 5) of commercial Relyx U200 (RU) and AllCem Core (AC) dual‐cure resin cements were injected in a cylindrical Teflon sampler (25 mm³) and separated according to polymerization mechanism: self‐cured (not irradiated) and dual‐cured (irradiated from the top surface with a LED device). The cement's volume was scanned with the μCT scanning conditions kept constant. To assess the depth dependence of polymerization contraction, it was measured the displacement of the cement mass from the sample holder at 30 vertical cuts (0.1 mm distant). To probe the microporosity, the percentage of area with presence of porosity by slice was obtained. All data were statistically treated. It was observed a positive linear correlation between depth and polymerization contraction in the irradiated groups. In the other hand, the concentration of micropores decreased with increasing depth. Furthermore, the composition of the resin cement was determinant for the correlation's coefficients of these physical properties with depth. The μCT technique showed to be useful to probe physical properties of dental restorative materials that influence in the clinical outcomes, revealing that, for thin specimens, when light cured the RU cement presented mechanical behavior more favorable for clinical applications.