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.     

Microscopy and microanalysis of crystalline glazes

Crystalline glazes on ceramic plates produced commercially in the U.K. and on ceramic pots produced commercially in Taiwan and Spain have been examined by X‐ray diffraction, conventional and polarized light microscopy, and scanning electron microscopy in order to identify the crystalline phases present in the glazes and to ascertain through X‐ray microanalysis the partitioning behaviour of the transition metal ions used to colour the glazes and the crystals within them. In each case examined, the macroscopic two‐dimensional spherulites within the glazes clearly seen by the naked eye were found to consist of large numbers of radially orientated acicular crystals each 5 µm or less in width embedded within the silica‐rich glaze. Energy dispersive X‐ray microanalysis and X‐ray diffraction of these crystals identified these crystals as willemite, α‐Zn₂SiO₄. The strong [001] texture of these crystals within the glaze evident from the X‐ray diffraction patterns was consistent with polarized light microscopy observations of the willemite crystals. In addition to willemite, small iron‐doped gahnite (ZnAl₂O₄) crystals were found in a honey‐coloured crystalline glaze and acicular rutile (TiO₂) crystals were found in the Portmeirion Pottery plates examined. Transition metal ions with a preference for tetrahedral coordination were observed to substitute for Zn²⁺ ions in willemite and to partition preferentially to the willemite crystals, whereas ions preferring octahedral coordination preferred to remain in the glaze.     

A technique for the examination of polar ice using the scanning electron microscope

The microstructure and location of impurities in polar ice are of great relevance to ice core studies. We describe a reliable method to examine ice in the scanning electron microscope (SEM). Specimens were cut in a cold room and could have their surfaces altered by sublimation either before (pre‐etching) or after (etching) introduction to the cryo‐chamber of the SEM. Pre‐etching was used to smooth surfaces, whilst etching stripped away layers from the specimen surface, aiding the location of particles in situ, and allowing embedded structures to be revealed. X‐ray analysis was used to determine the composition of localized impurities, which in some cases had been concentrated on the surface by etching. Examining uncoated surfaces was found to be advantageous and did not detract from qualitative X‐ray analysis. Imaging uncoated was performed at low accelerating voltages and probe currents to avoid problems of surface charging.     

Environmental chamber for in situ dynamic control of temperature and relative humidity during x-ray scattering

We have designed, constructed, and evaluated an environmental chamber that has in situ dynamic control of temperature (25 to 90?°C) and relative humidity (0% to 95%). The compact specimen chamber is designed for x-ray scattering in transmission with an escape angle of 2θ = ±30°. The specimen chamber is compatible with a completely evacuated system such as the Rigaku PSAXS system, in which the specimen chamber is placed inside a larger evacuated chamber (flight path). It is also compatible with x-ray systems consisting of evacuated flight tubes separated by small air gaps for sample placement. When attached to a linear motor (vertical displacement), the environmental chamber can access multiple sample positions. The temperature and relative humidity inside the specimen chamber are controlled by passing a mixture of dry and saturated gas through the chamber and by heating the chamber walls. Alternatively, the chamber can be used to control the gaseous environment without humidity. To illustrate the value of this apparatus, we have probed morphology transformations in Nafion(?) membranes and a polymerized ionic liquid as a function of relative humidity in nitrogen.     

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen‐binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti‐Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X‐ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA‐IgG anti Brucella biosensor was three‐fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 10⁶ CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody‐coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X‐ray photoelectron spectroscopy and confocal microscopy.     

Iodine vapor staining for atomic number contrast in backscattered electron and X‐ray imaging

Iodine imparts strong contrast to objects imaged with electrons and X‐rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation‐deposition state‐change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas‐tight container along with a small mass of solid I₂. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin‐embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X‐ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron‐ and photon‐sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.     

Is Scanning Electron Microscopy/Energy Dispersive X‐ray Spectrometry (SEM/EDS) Quantitative?

Scanning electron microscopy/energy dispersive X‐ray spectrometry (SEM/EDS) is a widely applied elemental microanalysis method capable of identifying and quantifying all elements in the periodic table except H, He, and Li. By following the “k‐ratio” (unknown/standard) measurement protocol development for electron‐excited wavelength dispersive spectrometry (WDS), SEM/EDS can achieve accuracy and precision equivalent to WDS and at substantially lower electron dose, even when severe X‐ray peak overlaps occur, provided sufficient counts are recorded. Achieving this level of performance is now much more practical with the advent of the high‐throughput silicon drift detector energy dispersive X‐ray spectrometer (SDD‐EDS). However, three measurement issues continue to diminish the impact of SEM/EDS: (1) In the qualitative analysis (i.e., element identification) that must precede quantitative analysis, at least some current and many legacy software systems are vulnerable to occasional misidentification of major constituent peaks, with the frequency of misidentifications rising significantly for minor and trace constituents. (2) The use of standardless analysis, which is subject to much broader systematic errors, leads to quantitative results that, while useful, do not have sufficient accuracy to solve critical problems, e.g. determining the formula of a compound. (3) EDS spectrometers have such a large volume of acceptance that apparently credible spectra can be obtained from specimens with complex topography that introduce uncontrolled geometric factors that modify X‐ray generation and propagation, resulting in very large systematic errors, often a factor of ten or more. SCANNING 35: 141‐168, 2013. ¹ Published 2012 Wiley Periodicals, Inc.     

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.     

Residual gas analysis in a SEM

It is shown that a clean (hydrocarbon partial pressure 0·1 times 10^?6 Pa) high vacuum (total pressure 5 times 10^?5 Pa) is obtainable in the specimen chamber of a scanning electron microscope by relatively inexpensive means.     

Potential of a sensitive uric acid biosensor fabricated using hydroxyapatite nanowire/reduced graphene oxide/gold nanoparticle

In this study, a ternary nanocomposite consisting of gold nanoparticles (AuNPs), hydroxyapatite (HAP) nanowires, and reduced graphene oxide (rGO) is synthesized by a simple one‐step hydrothermal method, which is used to modify glassy carbon electrode (GCE) for detecting uric acid. The nanocomposite is characterized through various methods such as scanning electron microscopy, transmission electron microscopy, and X‐ray diffraction. Electrochemical measurements of the modified GCE are performed in a conventional three‐electrode system. Experimental results show that the obtained HAP nanowire and rGO are mixed homogeneously, and the AuNPs are deposited into this matrix. The GCE modified by the nanocomposites have superior electrocatalytic activities for uric acid. The peak current intensities of UAO (uricase)/HAP‐rGO/AuNPs sensing system linearly increase as the uric acid concentration increases substantially in a range of 1.95 × 10^?5 to 6.0 × 10^?3 M (R² = .9943), with a detection limit of 3.9 × 10^?6 M (S/N = 3) and analytical sensitivity of 13.86 mA/M. The biosensor performs well in determining uric acid concentration in human urine samples.     

Development of a new analytical electron microscopy technique to quantify the chemistry of planar defects and to measure accurately solute segregation to grain boundaries

A new technique of analytical transmission electron microscopy called ConceptEM has been developed for determining highly accurately small amounts of solute or dopant atoms incorporated into well‐defined planar defects such as stacking faults, grain boundaries or interfaces. The method is based on recording series of analytical spectra taken with different electron beam diameters on the same position centred above a defect that is orientated either edge‐on or slightly inclined with respect to the electron beam. It can be applied to energy‐dispersive X‐ray spectroscopy or electron energy‐loss spectroscopy and necessitates only a nano‐probe modus but no scanning unit. Reliability and accuracy have been tested numerically under various conditions using simulations for a specific geometry, as a function of specimen thickness, material, acceleration voltage, collection angle, random beam displacements and solid solubility. The accuracy has been found to be substantially better (by factors of 5–10) than that of any other current standard technique based on single measurements. Our calculations suggest an accuracy in the determination of the Gibbsian solute excess at a special grain boundary down to ±1% of a monolayer, i.e. around ±0.1 atoms nm^?2 under typical experimental conditions, with a maximum error about twice as large. The parameter limiting a straightforward analysis is found to be the solid solubility, which itself, however, can be measured accurately by the technique so that it can be taken into account quantitatively and the above‐stated precision is retained.     

Reductive performance of ZVI/Cu polyscale particle to decolorize reactive black 5

Based on direct reducing of copper ions by commercially available zero valent iron (ZVI), ZVI/Cu polyscale particles with high activation are synthesized and exhibit significant removal efficiency for reactive black (RB) dye. The ZVI/Cu polyscale particles could maintain a good performance for RB removal at a wide pH range of 3.0–9.0. Different from the mineralization process of other organic pollutants in ZVI/Cu system, the degradation kinetics of RB could be described well by novel pseudo‐second kinetic models. Results of scanning electron microscopy (SEM), X‐ray fluorescence elemental analysis (EDX), and X‐ray diffraction (XRD) show the planting Cu has deposited on the surface of Fe⁰. The characterization of ZVI/Cu particles and RB solution before and after degradation reveals that decolorization of RB is not only resulted from the reduction process including direct reduction by accepting electrons from Fe⁰ oxidation and indirect reduction by atomic hydrogen generated on the ZVI/Cu surface, but also the oxidation of hydroxyl radicals in the mineralization procedure of RB dye.     

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.     

Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples

A new light microscope–temperature‐controlled chamber (LM–TCC) has been constructed. The special feature of the light microscope–temperature‐controlled chamber is the Peltier‐element temperature control of a specimen holder for biological samples, with a volume capacity of 1 mL. This system has marked advantages when compared to other approaches for temperature‐controlled microscopy. It works in a temperature range of −10°C to +95°C with an accuracy of ±0.1°C in the stationary phase. The light microscope–temperature‐controlled chamber allows rapid temperature shift rates. A maximum heating rate of 12.9°C min⁻¹ and a maximum cooling rate of 6.0°C min⁻¹ are achieved with minimized overshoots (≤1.9°C). This machinery operates at low cost and external coolants are not required. Especially with samples absorbing irradiation strongly, temperature control during microscopy is necessary to avoid overheating of samples. For example, leaf segments of Ficaria verna exposed to 4500 μmol photons m⁻² s⁻¹ in a standard microscopic preparation show a temperature increase (δT) of 18.0°C, whereas in the light microscope–temperature‐controlled chamber this is reduced to 4°C. The kinetics of microscope‐light induced δT are described and infrared thermography demonstrates the dissipation of the temperature. Chloroplasts of the cold adapted plant Ranunculus glacialis show the tendency to form stroma‐filled protrusions in relation to the exposure temperature. The relative number of chloroplasts with protrusions is reduced at 5°C when compared to 25°C. This effect is reversible. The new light microscope–temperature‐controlled chamber will be useful in a wide range of biological applications where a rapid change of temperature during microscopic observations is necessary or has to be avoided allowing a simulation of ecologically relevant temperature scenarios.     

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.     

Contact-free exhaust system for vacuum compatible gas bearing guides

Using linear gas bearing guides in a high vacuum environment, the common method to keep the vacuum quality is to exhaust the gas emitted by the bearing pads before leaking into the vacuum chamber. Thereby the exhaust tubes between the guide and the exhaust pumps should interfere with the guide as little as possible while maintaining a flexible connection and a highly effective exhaustion rate. A novel exhaust system that implements these requirements is described within this paper. The major achievement was the realization of two exhaust tubes slidable into one another combined with the known method of non-contact clearance seals, thus enabling an highly efficient and yet disturbance free exhaustion. This setup was developed and characterized at static and dynamic conditions. An analytical model for dimensioning the non-contact seal was worked out and experimentally verified. The number of seal stages and the clearance height were identified as the major impact factors on the leakage rate of the setup. It is concluded that the investigated approach is very suitable for vacuum compatible gas bearing guides since a vacuum level in the order of 10⁻⁴ Pa was maintained during the experiments.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

Electron backscatter diffraction applied to lithium sheets prepared by broad ion beam milling

Due to its very low hardness and atomic number, pure lithium cannot be prepared by conventional methods prior to scanning electron microscopy analysis. Here, we report on the characterization of pure lithium metallic sheets used as base electrodes in the lithium‐ion battery technology using electron backscatter diffraction (EBSD) and X‐ray microanalysis using energy dispersive spectroscopy (EDS) after the sheet surface was polished by broad argon ion milling (IM). No grinding and polishing were necessary to achieve the sufficiently damage free necessary for surface analysis. Based on EDS results the impurities could be characterized and EBSD revealed the microsctructure and microtexture of this material with accuracy. The beam damage and oxidation/hydration resulting from the intensive use of IM and the transfer of the sample into the microscope chamber was estimated to be <50 nm. Despite the fact that the IM process generates an increase of temperature at the specimen surface, it was assumed that the milling parameters were sufficient to minimize the heating effect on the surface temperature. However, a cryo‐stage should be used if available during milling to guaranty a heating artefact free surface after the milling process. Microsc. Res. Tech., 78:30?39, 2015. © 2014 Wiley Periodicals, 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.     

Optimizing use of the structural chemical analyser (variable pressure FESEM‐EDX raman spectroscopy) on micro‐size complex historical paintings characterization

The novel Structural Chemical Analyser (hyphenated Raman spectroscopy and scanning electron microscopy equipped with an X‐ray detector) is gaining popularity since it allows 3‐D morphological studies and elemental, molecular, structural and electronic analyses of a single complex micro‐sized sample without transfer between instruments. However, its full potential remains unexploited in painting heritage where simultaneous identification of inorganic and organic materials in paintings is critically yet unresolved. Despite benefits and drawbacks shown in literature, new challenges have to be faced analysing multifaceted paint specimens. SEM?Structural Chemical Analyser systems differ since they are fabricated ad hoc by request. As configuration influences the procedure to optimize analyses, likewise analytical protocols have to be designed ad hoc. This paper deals with the optimization of the analytical procedure of a Variable Pressure Field Emission scanning electron microscopy equipped with an X‐ray detector Raman spectroscopy system to analyse historical paint samples. We address essential parameters, technical challenges and limitations raised from analysing paint stratigraphies, archaeological samples and loose pigments. We show that accurate data interpretation requires comprehensive knowledge of factors affecting Raman spectra. We tackled: (i) the in‐FESEM?Raman spectroscopy analytical sequence, (ii) correlations between FESEM and Structural Chemical Analyser/laser analytical position, (iii) Raman signal intensity under different VP‐FESEM vacuum modes, (iv) carbon deposition on samples under FESEM low‐vacuum mode, (v) crystal nature and morphology, (vi) depth of focus and (vii) surface‐enhanced Raman scattering effect. We recommend careful planning of analysis strategies prior to research which, although time consuming, guarantees reliable results. The ultimate goal of this paper is to help to guide future users of a FESEM‐Structural Chemical Analyser system in order to increase applications.