High-resolution scanning electron microscopy of frozen-hydrated and freeze-substituted kidney tissue

Inner surfaces and fracture faces of rabbit kidney tissue were investigated with high-resolution scanning electron microscopy using two different cryopreparation techniques: (i) for the observation of fracture faces, cryofixed tissue was fractured and coated in a cryopreparation chamber dedicated to SEM, vacuum transferred onto a cold stage and observed in the frozen-hydrated state; (ii) for the observation of inner surfaces of the nephron, water was removed after freezing and fracturing by freeze substitution and critical-point drying of the tissue. By both methods, macromolecular structures such as intramembranous particles on fracture faces and particles on inner surfaces were imaged. The latter method was used to investigate in more detail surface structures of cells in the cortical collecting duct. These studies revealed a heterogeneity of intercalated cells not described thus far.     

Combined atomic force and scanning reflection interference contrast microscopy

A sphere attached to a cantilever is used simultaneously as an atomic force microscope (AFM) tip and as a curved reflective surface for producing scanning reflection interference contrast microscope (RICM) images of fluorescent beads dried onto a glass slide. The AFM and RICM images are acquired in direct registration which enables the identification of individually excited beads in the AFM images. The addition of a sharp, electron beam-deposited tip to the sphere gives nanometer resolution AFM images without loss of optical contrast.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Cross-sectional reflection electron microscopy of III–V compound epilayers

Reflection electron microscopy (REM) is shown to be applicable to the testing of the quality of Al_xGa_1?xAs/GaAs layer structures. Cross-sectional images of quantum well structures with layer thickness down to 1 nm can be obtained. The practical aspects of the REM technique are presented. The most important advantages and drawbacks of cross-sectional REM are discussed in detail.     

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.     

Silver-enhanced colloidal-gold labelling of rabbit kidney collecting-duct cell surfaces imaged by scanning electron microscopy

The luminal cell surfaces of rabbit kidney cortical collecting-duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20-μm-thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting-duct epithelium using biotinylated PNA and a 6-nm colloidal-gold-coupled antibody against biotin. Colloidal-gold labels were detected at low magnification (2000–4000 x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell-surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.     

Quantitative analysis of backscattered-electron contrast in scanning electron microscopy

Backscattered-electron scanning electron microscopy (BSE-SEM) imaging is a valuable technique for materials characterisation because it provides information about the homogeneity of the material in the analysed specimen and is therefore an important technique in modern electron microscopy. However, the information contained in BSE-SEM images is up to now rarely quantitatively evaluated. The main challenge of quantitative BSE-SEM imaging is to relate the measured BSE intensity to the backscattering coefficient η and the (average) atomic number Z to derive chemical information from the BSE-SEM image. We propose a quantitative BSE-SEM method, which is based on the comparison of Monte–Carlo (MC) simulated and measured BSE intensities acquired from wedge-shaped electron-transparent specimens with known thickness profile. The new method also includes measures to improve and validate the agreement of the MC simulations with experimental data. Two different challenging samples (ZnS/Zn(O_xS_1–x)/ZnO/Si-multilayer and PTB7/PC₇₁BM-multilayer systems) are quantitatively analysed, which demonstrates the validity of the proposed method and emphasises the importance of realistic MC simulations for quantitative BSE-SEM analysis. Moreover, MC simulations can be used to optimise the imaging parameters (electron energy, detection-angle range) in advance to avoid tedious experimental trial and error optimisation. Under optimised imaging conditions pre-determined by MC simulations, the BSE-SEM technique is capable of distinguishing materials with small composition differences.     

Morphological investigation of various orthodontic lingual bracket slots using scanning electron microscopy and atomic force microscopy

Various labial and lingual orthodontic appliances with aesthetic materials have been developed due to an increased demand in aesthetic orthodontic treatment. However, there are few reports regarding the morphology of lingual orthodontic appliances. Therefore, this study evaluates the roughness of slot surfaces of various orthodontic lingual brackets using field emission scanning electron microscopy (FE‐SEM) and atomic force microscopy (AFM). Three types of stainless steel lingual brackets (Stealth^®, 7th Generation^®, and Clippy L^®) and one gold lingual bracket (Incognito?) with a slot size of 0.018 inches × 0.025 inches (0.457 × 0.635 mm²) were selected as representative lingual materials. Both FE‐SEM and AFM examinations showed that the Stealth^® and Clippy L^® brackets had the lowest surface roughness, while the 7th Generation^® bracket had the highest surface roughness. There was a significant difference in surface morphology between the types of lingual brackets, even when composed of the same material. The surface roughness of the bracket slot was dependent on the manufacturing process or surface polishing process rather than the fundamental properties of the bracket materials. There was no significant difference in the mean surface roughness of the slot floor between gold and stainless steel lingual brackets. These findings suggest that, although the gold lingual bracket is very expensive, it has great potential for use in patients with nickel allergy.     

Silicon nitride windows for electron microscopy of whole cells

Silicon microchips with thin, electron transparent silicon nitride windows provide a sample support that accommodates both light‐, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and there is no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare the microchips for biological samples, and examples of images obtained using different light and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light and electron microscopy.     

Scanning reflection electron microscopy of surface topography by diffusely scattered electrons in the scanning electron microscope

In this paper, the technique of scanning reflection electron microscopy (SREM) by diffusely scattered electrons in the scanning electron microscope is described in detail. A qualitative account of the formation of image contrast in SREM is also described. It is assumed that, for grazing geometry, forward-scattered electrons reflect from regions close to the surface, following a few scattering events within the first few atomic layers, and lose very little energy in the process. The penetration depth of the primary electrons is very limited, resulting in strongly peaked envelopes of forward-scattered electrons. It is also assumed that a surface containing topographic features presents a range of tilt angles, resulting in different reflection coefficients. Tilt contrast results because each facet has a different scattering yield, which is dependent upon local surface inclination. Full details of the instrumentation designed for SREM are described, and to illustrate the technique, results recorded from an epitaxial GaAs on GaAs crystal, Pb₂(Zr,Ta)O₆ thin film on silicon, and SiO₂ amorphous film on silicon are presented.     

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.     

An anomalous contrast in scanning electron microscopy of insulators: the pseudo-mirror effect

In a scanning electron microscope, electron-beam irradiation of insulators may induce a strong electric field due to the trapping of charges within the specimen interaction volume. On one hand, this field modifies the trajectories of the beam of electrons subsequently entering the specimen, resulting in reduced penetration depth into the bulk specimen. On the other hand, it leads to the acceleration in the vacuum of the emitted secondary electrons (SE) and also to a strong distortion of their angular distribution. Among others, the consequences concern an anomalous contrast in the SE image. This contrast is due to the so-called pseudo-mirror effect. The aim of this work is first to report the observation of this anomalous contrast, then to give an explanation of this effect, and finally to discuss the factors affecting it. Practical consequences such as contrast interpretations will be highlighted.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Transmission electron microscopy staining methods for the cortex of human hair: a modified osmium method and comparison with other stains

For wool, superior staining of a wide range of ultrastructural components is achieved by en bloc treatment of fibres with a chemical reductant followed by osmium tetroxide. For human scalp hair, although staining quality is similar, the penetration of reagents is poor, resulting in large parts of the fibre cortex remaining unstained. Here we describe a modification to the reduction-osmication method in which reagents penetrate through a cut fibre end, allowing visualization of a wide range of features across the cortex. We compare the staining quality, artefacts and range of structure rendered visible using transmission electron microscopy for en bloc reduction-osmication to other staining alternatives including en bloc silver nitrate and section stains based on uranyl acetate and lead citrate, phosphotungstic acid, potassium permanganate, ammoniacal silver nitrate and some combinations of these stains. The effects of hair-care treatments are briefly examined.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

Cryogenic transmission electron microscopy study: preparation of vesicular dispersions by quenching microemulsions

We previously showed that long‐lived nanoemulsions, seeming initially vesicular, might be prepared simply by diluting and cooling (quenching) warm microemulsions with n‐hexadecane with precooled water. In this paper, we confirm that these systems are vesicular dispersions when fresh, and they can be made with similar structures and compositional dependence using alkanes with chain lengths ranging from octane to hexadecane. The nanostructures of fresh nanoemulsions are imaged with cryogenic transmission electron microscopy (cryo‐TEM). We confirm that water‐continuous microemulsions give simple dispersions of vesicles (sometimes unilamellar), typically less than 100 nm in diameter; these systems can avoid separation for over 2 months. Selected samples were also prepared using halogenated alkanes to create additional contrast in the cryo‐TEM, allowing us to confirm that the oil is located in the observed vesicular structures.     

Investigation of Si–Au vicinal surfaces using scanning tunnelling microscopy and reflection high-energy electron diffraction

Silicon vicinal surfaces can be successfully used as substrates for the preparation of one‐dimensional nanostructures. The quality of the structures prepared may be controlled using scanning tunnelling microscopy, as shown in this work. Additionally, it is possible to obtain valuable information using reflection high‐energy electron diffraction. A typical way of employing reflection high‐energy electron diffraction is to observe patterns of scattered electrons on a screen. However, it is possible to obtain more detailed information on the arrangement of atoms at the surface if azimuthal plots are collected. Azimuthal plots are measured by recording the intensity of specularly reflected electrons during the rotation of the sample around an axis perpendicular to its surface. So far, only flat surfaces have been examined in such a way. In this work, it is shown that such data, containing interesting features, can also be collected for vicinal surfaces.     

The effects of total internal reflection on the spread function along the axis in confocal microscopy

A broadening and splitting of the axial spread function is observed when high-numerical-aperture (NA) oil-immersion objectives are used on a confocal microscope to examine dielectric interfaces when the refractive index below the boundary is lower than the NA of the objective. The phenomena is due to total internal reflection probably as a consequence of the Goos-Hänchen shift. If total internal reflection occurs when undertaking confocal microscopy, this shift creates obvious problems when the optical sectioning capabilities must be optimal in reflectance mode and more subtle difficulties can arise when examining fluorescent emission. Alternatively, deliberately inducing total internal reflection can be used to estimate the refractive index in component parts within foams, emulsions and aerated specimens where such measurements can be relatively difficult to make by other means. Furthermore, the examination of total internal reflection with a confocal microscope permits the phenomena of total internal reflection itself to be probed with very high illumination intensities without disturbing the boundary conditions with an external probe. Finally, other changes in the apparent position of the focus were noted to occur when high-NA oil-immersion objectives are used to examine specimens such as metal mirrors.     

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.