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.     

Free-standing graphene by scanning transmission electron microscopy

Free-standing graphene sheets have been imaged by scanning transmission electron microscopy (STEM). We show that the discrete numbers of graphene layers enable an accurate calibration of STEM intensity to be performed over an extended thickness and with single atomic layer sensitivity. We have applied this calibration to carbon nanoparticles with complex structures. This leads to the direct and accurate measurement of the electron mean free path. Here, we demonstrate potentials using graphene sheets as a novel mass standard in STEM-based mass spectrometry.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Resolution in low voltage scanning electron microscopy

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.     

Improved preservation of bacterial exopolymers for scanning electron microscopy

Two methods of sample preparation, originally developed for transmission electron microscopy and better at preserving bacterial exopolymers than the traditional fixation with glutaraldehyde, were adapted for scanning electron microscopy. The first involves a glutaraldehyde-lysine mixture as a fixative. The second, based on the use of polycationic ferritin, was modified to include a post-fixation step with either glutaraldehyde or a glutaraldehyde-lysine mixture. These techniques were found to improve considerably the preservation of exopolymers secreted by three bacterial strains.     

A new rapid method of air-drying for scanning electron microscopy using tetramethylsilane

A new rapid air-drying technique which gives results comparable to critical point-drying is described for scanning electron microscopy, using a trematode parasite, Homalogaster paloniae, as a test specimen.     

Analytical scanning electron microscopy for solid surface

A scanning electron microscope of ultra-high-vacuum (UHV-SEM) with a field emission gun (FEG) is operated at the primary electron energies of from 100 eV to 3 keV. The instrument can form the images that contain information on surface chemical composition, chemical bonding state (electronic structure), and surface crystal structure in a microscopic resolution of several hundred angstroms (Å) using the techniques of scanning Auger electron microscope, scanning electron energy loss microscope, and scanning low-energy electron diffraction (LEED) microscope. A scanning tunneling microscope (STM) also has been combined with the SEM in order to obtain the atomic resolution for the solid surface. The instrumentation and examples of their applications are presented both for scanning LEED microscopy and STM.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

Prospects for lithium imaging using annular bright field scanning transmission electron microscopy: a theoretical study

There is strong interest in lithium imaging, particularly because of its significance in battery materials. However, light atoms only scatter electrons weakly and atomic resolution direct imaging of lithium has proven difficult. This paper explores theoretically the conditions under which lithium columns can be expected to be directly visible using annular bright field scanning transmission electron microscopy. A detailed discussion is given of the controllable parameters and the conditions most favourable for lithium imaging.     

A method for imaging single clay platelets by scanning electron microscopy

A method for preparing and observing clay platelets for size and shape analysis using scanning electron microscopy (SEM) was developed. Samples of the clay platelets were prepared by polyelectrolyte-assisted adsorption onto a pyrolytic graphite surface. The use of graphite as a substrate was advantageous because of the low number of secondary electrons emitted from it during imaging by SEM. The resulting low background noise allowed the emission from the approximately 1 nm thick clay sheets to be clearly visualized. Images of centrifuged montmorillonite showed large exfoliated platelets with lateral dimensions between 200 and 600 nm. In contrast, uncentrifuged montmorillonite appeared to contain a large amount of unexfoliated clusters. Although it was not possible to obtain high-quality images of the smaller sheets of Laponite RD, the images of this material did contain size features comparable to the approximately 30 nm2 size reported previously using light scattering, as well as transmission electron and atomic force microscopies.     

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.     

Tomography of asymmetric bulk specimens imaged by scanning electron microscopy

The scanning electron microscope produces nanometer-resolution surface images of biological samples preserved in a life-like state. Extracting three-dimensional information from these two-dimensional images has been the subject of long and ongoing research. We present here a general method and theoretical basis for reconstructing the surfaces of SEM specimens imaged from multiple directions by back-projection. The resulting reconstructions are faithful representations of the original specimen geometry, even when the input images are blurred and have low signal-to-noise ratio.     

Dynamics of annular bright field imaging in scanning transmission electron microscopy

We explore the dynamics of image formation in the so-called annular bright field mode in scanning transmission electron microscopy, whereby an annular detector is used with detector collection range lying within the cone of illumination, i.e. the bright field region. We show that this imaging mode allows us to reliably image both light and heavy columns over a range of thickness and defocus values, and we explain the contrast mechanisms involved. The role of probe and detector aperture sizes is considered, as is the sensitivity of the method to intercolumn spacing and local disorder.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.     

Internal medulla of hair revealed by scanning electron microscopy

The standard scanning electron microscope method for examining surface structure using the secondary electron mode has unexpectedly revealed the internal ladder-type medulla of some animal hairs.     

New system for secondary electron detection in variable-pressure scanning electron microscopy

A novel secondary electron detection system combining a two‐stage detector head and a differential pumping system is presented. The detector head consisted of a scintillation Everhart‐Thornley detector and a microsphere plate, separating it from the lower vacuum in the intermediate chamber (below 0.1 mbar). The system was arranged asymmetrically, which should contribute to a lower gas leakage through the plate and a longer life span of the plate. The system offered all the advantages of the scintillator detector in a wide range of gas pressures, from high vacuum to those of the order of 10 mbar, typical of high‐pressure scanning electron microscopy.     

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.