Characterization of lacquer films from the middle and late Chinese warring states period 476–221BC

The famous lacquer wares excavated from the Jiuliandun Tombs of the middle and late Warring States period (476–221 BC) were analyzed using scanning electron microscopy/energy as well as dispersive X‐ray spectrometry (SEM/EDS), Raman spectroscopy (RS), optical microscopy (OM), and X‐ray diffraction (XRD). The results showed a multilayer structure in the lacquer film, including a Qihui layer (a layer made of lacquer and various plasters), undercoat layer (or finishing coat) and colored paint layer mixed with various inorganic particles, such as quartz (SiO₂) and hydroxyapatite [Ca₅(PO₄)₃(OH)], as fillers in the Qihui layers or orpiment (As₂S₃) and cinnabar (HgS), which were used as a yellow or red pigment, respectively. With the help of elemental mapping images, a double‐layer structure of the lacquer plaster was observed, corresponding to a mixture of lacquer liquid and bone ash [Ca₅(PO₄)₃(OH)], with large‐diameter particles in the ground lacquer layer near the wooden body and small quartz (SiO₂) particles in upper lacquer layer. Specifically, quartz particles detected in the undercoat layer as fillers could be beneficial for improving the moshardness value, cost reduction and abrasive resistance of the lacquer film. In fact, the mixed method that used urushi and inorganic particles to form lacquer plaster was an important technological innovation and deeply influenced lacquering technologies worldwide. The results of this study will not only contribute to understanding the importance of lacquer skills in the Chinese Warring States but also provide information for cultural relic conservation as well as modern lacquer manufacturing for their protection and duplication.     

Morphology,structure and chemistry of extracted diesel soot: Part II: X-ray absorption near edge structure (XANES) spectroscopy and high resolution transmission electron microscopy

Chemical composition of diesel soot extracted from used diesel engine has been investigated using X-ray Absorption Near Edge Structure (XANES) spectroscopy and high-resolution transmission electron microscopy (HRTEM). The XANES spectra indicate the presence of phosphates of zinc and calcium and sulfates of zinc and calcium as well as the presence of sulfides of zinc. HRTEM coupled with EDS and lattice imaging indicates the presence of nanoparticles of Ca₃(PO₄)₂ and Fe₂O₃ embedded in the turbostratic soot structure. Both Ca₃(PO₄)₂ and Fe₂O₃ are hard particles and appear to have been incorporated within the turbostratic soot structure during third body wear.     

Theoretical explanation of the relationship between backscattered electron and x-ray linear attenuation coefficients in calcified tissues

X-ray absorption and backscattered electron (BSE) microscopies are two commonly used techniques for estimating mineral contents in calcified tissues. The resolution in BSE images is usually higher than in x-ray images, but due to the previous lack of good standards to quantify the grey levels in BSE images of bones and teeth, x-ray microtomog-raphy (XMT) images of the same specimens have been used for calibration. However, the physics of these two techniques is different: for a specimen with a given composition, the x-ray linear attenuation coefficient is proportional to density, but there is no such relation with the BSE coefficient. To understand the reason that this calibration appears to be valid, the behaviour of simulated bone samples was investigated. In this, the bone samples were modelled as having three phases: hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), protein, and void (either empty or completely filled with polymethylmethacrylate (PMMA), a resin which is usually used for embedding bones and teeth in microscopic studies). The x-ray linear attenuation coefficients (calculated using published data) and the BSE coefficients (calculated using Monte Carlo simulation) were compared for samples of various phase proportions. It was found that the BSE coefficient correlated only with the x-ray attenuation coefficient for samples with PMMA infiltration. This was attributed to the properties of PMMA (density and mean atomic number) being very similar to those of the protein; therefore, the sample behaves like a two-phase system which allows the establishment of a monotonic relation between density and BSE coefficient. With the newly developed standards (brominated and iodinated dimethacrylate esters) for BSE microscopy of bone, grey levels can be converted to absolute BSE coefficients by linear interpolation, from which equivalent densities can be determined.     

Study of two tungstates Ca0.5Cd0.5WO4 and Ca0.2Cd0.8WO4 by transmission electron microscopy

To better understand the role of crystal structures and local disorder in the photonic properties of the system (1 ? x)CaWO₄ ? xCdWO₄ with 0 < x < 1, two specific phases with compositions x = 0.5 (scheelite phase) and 0.8 (wolframite phase) have been studied by scanning and transmission electron microscopies. High‐resolution electron microscopy images and image simulations, associated with X‐ray diffraction data, allowed confirming the lattices and space groups I4₁/a and P2/c of the two scheelite and wolframite phases, at the local scale. The electron microscopy data show the existence of a high degree of crystallization associated with statistical distribution of Ca or Cd atoms on a Ca_1?xCd_x site in each lattice.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Experimental ionization cross-sections of phosphorus and calcium by electron spectroscopic imaging

The absolute partial electron scattering cross-section for the phosphorus L_2,3-shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross-section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂. At 80kcV, the partial cross-section of phosphorus was 2.26 times 10^?20 and 2.68 × 10^?20cm²/atom for poliovirus and hydroxyapatite, respectively, at 150eV loss for a 15-cV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross-section was 0.49 × 10^?20 cm²/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross-sections calculated either by hydrogenic or Hartree—Slater approaches.     

Recent developments and new strategies in scanning electron microscopy*

In addition to improvements in lateral resolution in scanning electron microscopy, recent developments of interest here concern extension of the incident beam energy, E₀, over two decades, from ≈ 20 keV to ≈ 0.1–0.5 keV and the possibility of changing the take-off emission, α, of detected secondary electrons. These two degrees of freedom for image acquisition permit a series of images of the same field of view of a specimen to be obtained, each image of the series differing from the others in some aspect. The origins of these differences are explored in detail and they are tentatively interpreted in terms of the change in the secondary electron emission yield δ vs. E₀, δ = f(E₀), and also of the change in δ vs. α, ∂δ/∂α. Various origins for the chemical contrast and topographic contrast have been identified. Illustrated by correlating a secondary electron image and a backscattered electron image, use of the scatter diagram technique facilitates image comparison. The difference between the lateral resolution and the size of the minimum detectable detail is outlined to avoid possible errors in nanometrology. Some aspects related to charging are also considered and possible causes of contrast reversal are suggested. Finally, the suggested strategy consists of the acquisition of various images of a given specimen by changing one parameter: primary beam energy and take-off angle for conductive specimens; working distance or beam intensity for high-resolution experiments; scanning frequency for insulating specimens.     

Essential experimental parameters for quantitative structure analysis using spherical aberration-corrected HAADF-STEM

The accuracy of quantitative analysis for Z-contrast images with a spherical aberration (C_s) corrected high-angle annular dark-field (HAADF) scanning transmission electron microscope (STEM) using SrTiO₃(0 0 1) was systematically investigated. Atomic column and background intensities were measured accurately from the experimental HAADF-STEM images obtained under exact experimental condition. We examined atomic intensity ratio dependence on experimental conditions such as defocus, convergent semi-angles, specimen thicknesses and digitalized STEM image acquisition system: brightness and contrast. In order to carry out quantitative analysis of C_s-corrected HAADF-STEM, it is essential to determine defocus, to measure specimen thickness and to fix setting of brightness, contrast and probe current. To confirm the validity and accuracy of the experimental results, we compared experimental and HAADF-STEM calculations based on the Bloch wave method.     

Pressure-induced amorphization of zinc orthophosphate—Insight in the zinc coordination by XAS

In-situ high pressure X-ray absorption spectroscopy measurements at Zn K-edge were performed on α-Zn₃(PO₄)₂ compound, one of the model materials of the tribochemical film obtained from the ZDDP molecule. The goal of these synchrotron experiments was to investigate pressure-induced effects on the local environment of zinc atoms during a compression/decompression cycle. Modifications of the absorption spectra at Zn K-edge of α-Zn₃(PO₄)₂ show irreversible amorphization around zinc atoms and irreversible increase of the Zn–O distance. These results were identified as the signature of structural changes around the Zn atom in the pressed sample, thus suggesting an irreversible Zn coordination change with the pressure.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Transient analysis of gaseous electron-ion recombination in the environmental scanning electron microscope

Most of the work carried out in relation to contrast mechanisms and signal formation in an environmental scanning electron microscope has yet to consider the time dependent aspects of image generation at a quantitative level. This paper quantitatively describes gaseous electron‐ion recombination (also known as ‘signal scavenging’) in an environmental scanning electron microscope at a transient level by utilizing the dark shadows/streaks seen in gaseous secondary electron detector images of alumina (Al₂O₃) immediately after a region of enhanced secondary electron emission is encountered by a scanning electron beam. The investigation firstly derives a theoretical model of gaseous electron‐ion recombination that takes into consideration transients caused by the time constant of the gaseous secondary electron detector electronics and external circuitry used to generate images. Experimental data of pixel intensity versus time of the streaks are then simulated using the model enabling the relative magnitudes of (i) ionization and recombination rates, (ii) recombination coefficients and (iii) electron drift velocities, as well as absolute values of the total time constant of the gaseous secondary electron detection system and external circuitry, to be determined as a function of microscope operating parameters such as gaseous secondary electron detector bias, sample‐electrode separation, imaging gas pressure, and scan speed. The results revealed, for the first time, the exact dependence that the effects of secondary electron‐ion recombination on signal formation has on reduced electric field and time in an environmental scanning electron microscope. Furthermore, the model implicitly demonstrated that signal loss as a consequence of field retardation due to ion space charges, although obviously present, is not the foremost phenomenon causing streaking in images, as previously thought.     

MRT letter: Extended depth from focus reconstruction method for stretch zone measurement in 15-5PH steel

The stretch zone width (SZW) data for 15‐5PH steel CTOD specimens fractured at ?150°C to + 23°C temperature were measured based on focused images and 3D maps obtained by extended depth‐of‐field reconstruction from light microscopy (LM) image stacks. This LM‐based method, with a larger lateral resolution, seems to be as effective for quantitative analysis of SZW as scanning electron microscopy (SEM) or confocal scanning laser microscopy (CSLM), permitting to clearly identify stretch zone boundaries. Despite the worst sharpness of focused images, a robust linear correlation was established to fracture toughness (K_C) and SZW data for the 15‐5PH steel tested specimens, measured at their center region. The method is an alternative to evaluate the boundaries of stretched zones, at a lower cost of implementation and training, since topographic data from elevation maps can be associated with reconstructed image, which summarizes the original contrast and brightness information. Finally, the extended depth‐of‐field method is presented here as a valuable tool for failure analysis, as a cheaper alternative to investigate rough surfaces or fracture, compared to scanning electron or confocal light microscopes. Microsc. Res. Tech. 75:1155–1158, 2012. © 2012 Wiley Periodicals, Inc.     

Image simulation of high resolution energy filtered TEM images

Inelastic image simulation software is presented, implementing the double channeling approximation which takes into account the combination of multiple elastic and single inelastic scattering in a crystal. The approach is described with a density matrix formalism. Two applications in high resolution energy filtered (EFTEM) transmission electron microscopy (TEM) images are presented: thickness-defocus maps for _SrTiO3${\mathrm{SrTiO}}_3$ and exit plane intensities for an (_LaAlO3)3(_SrTiO3)3$({\mathrm{LaAlO}}_3{)}_3({\mathrm{SrTiO}}_3{)}_3$ multilayer system. Both systems show a severe breakdown in direct interpretability which becomes worse for higher acceleration voltages, thicker samples and lower excitation edge energies. Since this effect already occurs in the exit plane intensity, it is a fundamental limit and image simulations in EFTEM are indispensable just as they are indispensable for elastic high resolution TEM images.     

The observation of ferroelastic/ferroelectric domains in synthetic Mn3B7O13X single-crystal boracites by light and electron microscopy

Crystals of halogen manganese boracite, the mineral Mg₃B₇O₁₃Cl, which is currently found in bedded sedimentary deposits of anhydrite, gypsum and halite, have been grown by chemical transport reactions and were examined by polarizing light microscopy and scanning electron microscopy. For both methods the same as-grown samples were used without having to metallize the crystal faces. Comparative electron microscope images were useful not only for observing the charging mechanism of an insulating sample bombarded by an electron beam but also for revealing the domain structure of these ferroelectric/ferroelastic materials previously observed between the crossed polars of a light microscope. EDS qualitative analysis of the crystal faces was performed for the three compositions under study, i.e. Mn₃B₇O₁₃Cl, Mn₃B₇O₁₃Br and Mn₃B₇O₁₃I.     

Comparison of manual and automatic processing of biological samples for electron microscopy

The ultrastructure of a sample can be observed by electron microscopy (EM), which has become an indispensable research tool in morphological studies. However, EM sample preparation techniques are complicated and time‐consuming, with a high labor cost. The current study was conducted to compare the conventional manual and automated methods for sample processing and post‐staining for electron microscopy. Automated sample processing reduces OsO₄ contamination, improves the efficiency of sample preparation and is easy to use. Therefore, the results of their study provide a practical and feasible method for the preparation of biological samples for electron microscopy.     

Elemental composition of pyroantimonate precipitates analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS) in vitellogenic ovarian follicles of Drosophila

Ca²⁺ was precipitated with potassium antimonate in vitellogenic follicles of the fruit fly Drosophila melanogaster and the distribution of the precipitates formed was studied by electron microscopy. The microvilli of the oolemma in mid- and late vitellogenic follicles were lined with precipitates. The chemical composition of the precipitates was analysed by electron spectroscopic imaging (ESI). The images produced by inelastically scattered electrons at specific ionization edges were compared, and the nonspecific background signals were subtracted by an image processing system. The presence of Ca²⁺, antimony and oxygen in the precipitates formed could be demonstrated. The elemental composition of the precipitates and of yolk spheres was also analysed by electron energy-loss spectroscopy (EELS). With respect to the precipitates, signals at the calcium L_2,3-edge, the oxygen K-edge and the antimony M_4,5-edge were recorded without deconvolution and background subtraction. The yolk spheres, which were free of precipitates, gave the characteristic signal of the nitrogen K-edge. The applied techniques combine good ultrastructural resolution with the possibility of analysing the elemental composition of histochemical reaction products and cellular structures.     

Electron-beam-induced fabrication of metal-containing nanostructures

An experimental system based on a transmission electron microscope JEM-100CX has been developed for electron beam-induced chemical vapor deposition. Direct electron beam-induced growth of nanometer-wide self-supporting rods has been performed inside the microscope operating in scanning mode by decomposition of carbonyls of chromium Cr(CO)₆, tungsten W(CO)₆, and rhenium Re₂(CO)₁₀. In situ phase and structure transformations under annealing inside the microscope column were studied. Nanoscale rods and strips grown from rhenium carbonyl are of special interest because, after annealing, they consist of a single pure rhenium phase. The described method of metallic nanoelements fabrication enables us to produce highly conductive nanowires and tips for application in nanoelectronics, emission electronics, and scanning tunneling microscopy.     

Image quality evaluation for FIB-SEM images

Focused ion beam scanning electron microscopy (FIB-SEM) tomography is a serial sectioning technique where an FIB mills off slices from the material sample that is being analysed. After every slicing, an SEM image is taken showing the newly exposed layer of the sample. By combining all slices in a stack, a 3D image of the material is generated. However, specific artefacts caused by the imaging technique distort the images, hampering the morphological analysis of the structure. Typical quality problems in microscopy imaging are noise and lack of contrast or focus. Moreover, specific artefacts are caused by the FIB milling, namely, curtaining and charging artefacts. We propose quality indices for the evaluation of the quality of FIB-SEM data sets. The indices are validated on real and experimental data of different structures and materials.     

Traceable calibration of transfer standards for scanning probe microscopy

A Metrological Atomic Force Microscope (MAFM) has been constructed for the traceable calibration of transfer standards for scanning probe microscopy. It uses optical interferometry to generate image scales with direct traceability to the national standard of length. Three interferometers monitor the relative displacements of the AFM tip and sample in the x, y and z directions and the interferometer data is used directly to construct 3D images of sample surfaces. Traceable dimensional measurement of surface features may then be derived from the image data. This paper describes the MAFM instrument and presents a measurement uncertainty budget. Examples are given of measurements of pitch and step height on calibration transfer standards for scanning probe microscopy.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.