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.     

X‐ray diffraction of amorphous and crystalline overbased sulphonates

Colloidal dispersions of calcium carbonate stabilised by overbased sulphonates are used as lubricant additives to provide detergency, remove acidity, and prevent wear. In the present work various overbased sulphonate compounds have been analysed using X‐ray diffraction (XRD). The amorphous overbased sulphonates studied displayed broad diffraction patterns while the crystalline overbased sulphonates gave well‐defined diffraction patterns, correlating with the particle size. The XRD distances of the series of Mg, Ca, and Ba overbased sulphonates shifted to greater values and displayed a clear correlation (r = 0.98) with the ionic radius of the cations used.     

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.     

Energy dispersive X‐ray spectrometry study of the protective effects of fluoride varnish and gel on enamel erosion

Background: Dental erosion is a risk factor for dental health, introduced by today's lifestyle. Topical fluoride applications in the form of varnishes and gel may lead to deposition of fluoride on enamel. Purpose: This in vitro study aimed to evaluate the effect of two fluoride varnishes and one fluoride gel on the dissolution of bovine enamel by acids. Methods: Enamel samples (72) were divided (n = 8): artificial saliva (control‐G1), Pepsi Twist® (G2), orange juice (G3), Duraphat® + Pepsi Twist® (G4), Duraphat® + orange juice (G5), Duofluorid® + Pepsi Twist® (G6), Duofluorid® + orange juice (G7), fluoride gel + Pepsi Twist® (G8), and fluoride gel + orange juice (G9). Fluoride gel was applied for 4 min and the varnishes were applied and removed after 6 h. The samples were submitted to six cycles (demineralization: Pepsi Twist® or orange juice, 10 min; remineralization: saliva, 1 h). Samples were analyzed by energy‐dispersive X‐ray fluorescence (144 line‐scanning). Results: The amount of Ca and P decreased significantly in the samples of G2 and G3, and the Ca/P ratio decreased in G3. Mineral gain (Ca) was greater in G9 samples than in G4 > G3 > G5 > G1, and (P) greater in G7 samples than in G9 > G4‐6 > G2‐3. Conclusions: The protective effect of Duofluorid® was significantly lower than fluoride gel against orange juice. The fluoride varnishes can interfere positively with the dissolution of dental enamel in the presence of acidic beverages. Fluoride gel showed the best protection level to extrinsic erosion with low costs. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Application of sensitive,high‐resolution imaging at a commercial lab‐based X‐ray micro‐CT system using propagation‐based phase retrieval

Several dedicated commercial lab‐based micro‐computed tomography (μCT) systems exist, which provide high‐resolution images of samples, with the capability to also deliver in‐line phase contrast. X‐ray phase contrast is particularly beneficial when visualizing very small features and weakly absorbing samples. The raw measured projections will include both phase and absorption effects. Extending our previous work that addressed the optimization of experimental conditions at the commercial ZEISS Xradia 500 Versa system, single‐distance phase‐contrast imaging is demonstrated on complex biological and material samples. From data captured at this system, we demonstrate extraction of the phase signal or the correction of the mixed image for the phase shift, and show how this procedure increases the contrast and removes artefacts. These high‐quality images, measured without the use of a synchrotron X‐ray source, demonstrate that highly sensitive, micrometre‐resolution imaging of 3D volumes is widely accessible using commercially advanced laboratory devices.     

Evaluation of 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.     

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.     

X‐ray CT and histological imaging of xylem vessels organization in Mimosa pudica

Mimosa pudica has three distinct specialized organs, namely, pulvinus, secondary pulvinus, and pulvinule, which are respectively controlling the movements of petioles, leaflets, and pinna in response to external stimuli. Water flow is a key factor for such movements, but detailed studies on the organization of the vascular system for water transport in these organs have not been published yet. In this study, organizations of the xylem vessels and morphological features of the pulvinus, the secondary pulvinus, and the pulvinule were experimentally investigated by X‐ray computed tomography and histological technique. Results showed that the xylem vessels were circularly distributed in the specialized motile organs and reorganized into distinct vascular bundles at the extremities. The number and the total cross‐sectional area of the xylem vessels were increased inside the specialized motile organs. Morphological characteristics obtained in this study provided new insight to understand the functions of the vascular networks in the dynamic movements of M. pudica. Microsc. Res. Tech. 76:1204–1212, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Application of X‐ray microcomputed tomography in the characterization of irradiated nuclear fuel and material specimens

X‐ray microcomputed tomography (μCT) was applied in characterizing the internal structures of a number of irradiated materials, including carbon‐carbon fibre composites, nuclear‐grade graphite and tristructural isotropic‐coated fuel particles. Local cracks in carbon‐carbon fibre composites associated with their synthesis process were observed with μCT without any destructive sample preparation. Pore analysis of graphite samples was performed quantitatively, and qualitative analysis of pore distribution was accomplished. It was also shown that high‐resolution μCT can be used to probe internal layer defects of tristructural isotropic‐coated fuel particles to elucidate the resulting high release of radioisotopes. Layer defects of sizes ranging from 1 to 5 μm and up could be isolated by tomography. As an added advantage, μCT could also be used to identify regions with high densities of radioisotopes to determine the proper plane and orientation of particle mounting for further analytical characterization, such as materialographic sectioning followed by optical and electron microscopy. In fully ceramic matrix fuel forms, despite the highly absorbing matrix, characterization of tristructural isotropic‐coated particles embedded in a silicon carbide matrix was accomplished using μCT and related advanced image analysis techniques.     

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.     

Development and comparison of the methods for quantitative electron probe X‐ray microanalysis analysis of thin specimens and their application to biological material

In recent years, there has been a return to the use of electron probe X‐ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff‐Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff‐Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin‐embedded materials emphasized.     

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.     

Atomic force microscopy and energy dispersive X‐ray spectrophotometry analysis of reciprocating and continuous rotary nickel‐titanium instruments following root canal retreatment

The aim was to examine the effect of retreatment process on the surface roughness and nickel titanium (NiTi) composition of ProTaper Universal Retreatment (PTUR; consists of 3 files; D1, D2, D3) and WaveOne Gold (WOG) (primary) instruments. Twenty extracted mandibular molar teeth with severe curved (30–40°) mesial roots were selected and divided into two groups (n = 10) based on the instrument used for the removal of the root canal filling. Before and after using the instruments in two canals, they were subjected to atomic force microscopy (AFM) and energy dispersive X‐ray spectrophotometry (EDX) analysis. The EDX analysis data and roughness average (Ra) and root mean square (RMS) values were analyzed statistically using a one‐way analysis of variance and post hoc Tukey's test at the 5% significant level. There was no significant difference between the new and used D1 and D2 PTUR and WOG instruments in terms of the Ni composition (p > .05). The Ti contents of the used D2 and D3 PTUR instruments were lower those of the new instruments (p < .05). In both new and used instruments, PTUR and WOG have no difference in terms of Ra and RMS values. (p > .05). The Ra and RMS values of the PTUR and WOG systems significantly increased after removal of the root canal filling (p < .05). The use of PTUR and WOG instruments for removal of root canal filling in severely curved root canals affected the surface topography of the files. The NiTi composition of the WOG instruments was unaffected by the retreatment process.     

Adverse effects of respiratory disease medicaments and tooth brushing on teeth: A scanning electron microscopy,X‐ray fluorescence and infrared spectroscopy study

The present study aims to evaluate the effect of brushing with fluoride dentifrice on teeth severely affected by erosion due to respiratory medicaments. Enamel (n = 50) and dentin (n = 50) bovine specimens were prepared and treated with artificial saliva (S‐control), acebrofilin hydrochloride (AC), ambroxol hydrochloride (AM), bromhexine hydrochloride (BR), and salbutamol sulfate (SS) and subjected to cycles of demineralization (immersing in 3 mL, 1 min, three times a day at intervals of 1 hr, for 5 days) followed by remineralization (saliva, 37°C, 1 hr). Simulated brushing with fluoridated toothpaste was performed using 810 strokes in a reciprocal‐action brushing simulator. Scanning electron microscopy, micro energy dispersive X‐ray fluorescence (μ‐EDXRF) spectroscopy and attenuated total reflection Fourier transform infrared (ATR FTIR) spectroscopy were then performed. μ‐EDXRF images showed extensive erosion after treatment with all medicaments. SEM images showed enamel erosion in order SS > BR > AC = AM > S after brushing and fluoridation. FTIR results were in agreement. In case of dentin, μ‐EDXRF measurements showed significant difference in mineral content (percent weight of calcium and phosphate) in SS + brushing + fluoridation treated enamel compared to control, while μ‐EDXRF images showed erosive effects in the order SS > AM>BR > AC = S post brushing + fluoridation. SEM images showed erosion in the order SS > AM = BR > AC > S post brushing + fluoridation. Again, FTIR multivariate results were in agreement. Overall, our study shows that proper oral care is critical when taking certain medication. The study also demonstrates the possible use of FTIR for rapid clinical monitoring of tooth erosion in clinics.