Environmental scanning electron microscopy of marine aggregates

Marine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy-dispersive X-ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

Manipulating biological samples for environmental scanning electron microscopy observation

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.     

Environmental scanning electron microscopy observation of the ultrastructure of Demodex

In this study, numbers of Demodex of hair follicles and sebaceous glands were prepared and the ultrastructure (especially the mouthparts) of Demodex was observed firstly with environmental scanning electron microscopy (ESEM). The most suitable treatment methods and optimal environmental condition for observing the genus samples were found. The samples were washed with detergent and rinsed with distilled water, and then were taken to the specimen stage, on which there was carbon adhesive tape, using special tools. When the temperature was at 5 degrees C and chamber pressure at 5 mbar respectively, the surface of the samples could be fully imaged without covering water or dehydration. The sample surfaces were plump and clear without postmortem changes and charging artifacts. Detailed information about each part of Demodex was observed by ESEM, and clear three-dimensional images were recorded. The mouthparts of D. folliculorum were composed of a complex set of structures, which included a round oral opening, a sharp oral needle, and a special hypostome that looked like a longitudinal spindle in the central position. On the end segment of palpus, there were seven strong palpal claws located on each side of the mouthparts. D. folliculorum had special piercing mouthparts, while the mouthparts of D. brevis were a simpler structure. We could not observe the oral needle of D. brevis, and there were only five pairs of palpal claws on the end segment of palpus. The offensive organs of Demodex resulted in its pathogenic effects. After studying hundreds of Demodex, we identified both female and male species of D. folliculorum, but only females of D. brevis in our sample.     

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.     

Simple modifications for accurate high-temperature gas exposures using scanning electron microscopy

Relatively low-cost modifications to standard commercial scanning electron microscopes (SEM) that allow accurate exposure of sample(s) to noncorrosive gases at ambient and high temperatures are outlined. Energy-dispersive spectroscopic analysis of sample(s) exposed to noncorrosive gases at high temperatures is demonstrated. Gas exposure is limited to pressures of less than 10^?4 torr (1.33 × 10^?2 Pa) as a result of limitations on SEM system operation and SEM safety interlocks. Gases are limited to noncorrosive types as a result of potential damage to system detection devices and internal mechanical parts.     

Environmental scanning electron microscopy of personal and household products

The ability to forego sample preparation and to make observations directly in the environmental scanning electron microscope has benefited both household and personal product research at Unilever Research. Product efficacy on biological materials such as microcomedones was easily ascertained. Skin biopsies were examined in a moist state with no sample preparation. Effects of relative humidity on detergents were visually determined by recreating the necessary conditions in the microscope. Effects of cooling rates on the morphology of softener sheet actives that remained on polyester fabric were characterized via dynamic experimentation.     

Charging in scanning electron microscopy "from inside and outside"

This paper is an attempt to analyse most of the complicated mechanisms involved in charging and discharging of insulators investigated by scanning electron microscopy (SEM). Fundamental concepts on the secondary electron emission (SEE) yield from insulators combined with electrostatics arguments permit to reconsider, first, the widespread opinion following which charging is minimised when the incident beam energy E0 is chosen to be equal to the critical energy E(o)2, where the nominal total yield delta(o) + eta(o) = 1. For bare insulators submitted to a defocused irradiation, it is suggested here that the critical energy under permanent irradiation EC2 corresponds to a range of primary electrons, R, and nearly equals the maximum escape depth of the secondary electrons, r. This suggestion is supported by a comparison between published data of the SEE yield delta(o) of insulators (short pulse experiments) and experimental results obtained from a permanent irradiation for EC2. New SEE effects are also predicted at the early beginning of irradiation when finely focused probes are used. Practical considerations are also developed, with specific attention given to the role of a contamination layer where a negative charging may occur at any beam energy. The role of the various time constants involved in charging and discharging is also investigated, with special attention given to the dielectric time constant, which explains the dose rate-dependent effects on the effective landing energy in the steady state. Numerical applications permit to give orders of magnitude of various effects, and several other practical consequences are deduced and illustrated. Some new mechanisms for the contrast reversal during irradiation or with the change of the primary electron (PE) energy are also suggested.     

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.     

An environmental scanning electron microscopy study of aqueous gibbsite suspensions

Synthetic gibbsite has been used as a model system for study in the environmental scanning electron microscopy (ESEM), to probe its utility as a tool to study clay dispersions under different conditions of aggregation. We have been able to show that we can study the nature of the platelet interactions as the pH is altered, by imaging the dispersions after water evaporation from the surface to permit the surface of the platelets to be clearly seen. It has been possible to show that at alkaline pH there are very few face-edge contacts between the platelets, consistent with what is known about the charges at high pH on the faces and edges of the gibbsite. In contrast, at lower pHs, when faces and edges have opposite sign charges, there are significantly more platelets touching with edge and faces in contact. Finally, when the salt lithium chloride is added to a dispersion at approximately neutral pH, the plates appear to stack suggesting face-face interactions in the dispersion. Thus, ESEM has been able to demonstrate the variability of packing in gibbsite dispersions and to correlate the structures observed with the known charge distribution on the gibbsite platelets.     

The fine structure of fenestrated adrenocortical capillaries revealed by in-lens field-emission scanning electron microscopy and scanning transmission electron microscopy

Cell biologists probing the physiologic movement of macromolecules and solutes across the fenestrated microvascular endothelial cell have used electron microscopy to locate the postulated pore within the fenestrae. Prior to the advent of in-lens field-emission high-resolution scanning electron microscopy (HRSEM) and ultrathin m et al coating technology, quick-freeze, platinum-carbon replica and grazing thin-section transmission electron microscopy (TEM) methods provided two-dimensional or indirect imaging methods. Wedge-shaped octagonal channels composed of fibrils interwoven in a central mesh were depicted as the filtering structures of fenestral diaphragms in images of platinum replicas enhanced by photographic augmentation. However, image accuracy was limited to replication of the cell surface. Subsequent to this, HRSEM technology was developed and provided a high-fidelity, three-dimensional topographic image of the fenestral surface directly from a fixed and dried bulk adrenal specimen coated with a 1 nm chromium film. First described from TEM replicas, the “flower-like” structure comprising the fenestral pores was readily visualized by HRSEM. High-resolution images contained particulate ectodomains on the lumenal surface of the endothelial cell membrane. Particles arranged in a rough octagonal shape formed the fenestral rim. Digital acquisition of analog photographic recordings revealed a filamentous meshwork in the diaphragm, thus confirming and extending observations from replica and grazing section TEM preparations. Endothelial cell pockets, first described in murine renal peritubular capillaries, were observed in rhesus and rabbit adrenocortical capillaries. This report features recent observations of fenestral diaphragms and endothelial pockets fitted with multiple diaphragms utilizing a Schottky field-emission electron microscope. In-lens staging of bulk and thin section specimens allowed tandem imaging in HRSEM and scanning TEM modes at 25 kV.     

Dinosaur eggshell study using scanning electron microscopy

Visualization and analysis of structural features in fossil dinosaur eggs by scanning electron microscopy augment information from traditional petrographic light microscopy. Comparison of characteristics in fossil and modern eggshells allows inferences to be made regarding dinosaur reproductive biology, physiology, and evolutionary relationships. Assessment of diagenetic alteration of primary eggshell calcite structure that occurs during fossilization provides important information necessary for taxonomic identification and paleoenvironmental interpretations.     

Dynamic secondary electron contrast effects in liquid systems studied by environmental scanning electron microscopy

We report an investigation into a dynamic contrast phenomenon in water-oil emulsions imaged in the environmental scanning electron microscope. Secondary electron contrast between oil and water phases is shown to change with scan rate, even inverting in extreme cases. This effect is attributed to the fact that charge carriers in liquids have intermediate mobilities compared with those in metallic conductors and solid insulators. Thus, increasing the electron energy flux density (via slower scan rates) results in the temporary accumulation of excess charge, which in turn gives rise to increased secondary electron emission. Excess charge dissipates between frames, however, such that classical charging of the specimen is not observed. The oils used here have conductivities lower than that of water, making them more susceptible to the effect. However, the material within the primary electron interaction volume is a conductive medium. We demonstrate that charging effects are not seen in regions of the oil where the interaction volume is in contact with the more conductive continuous water phase. Secondary electron emission from these regions therefore approximates to the intrinsic yield.     

Low voltage scanning electron microscopy

The scanning electron microscope (SEM) is usually operated with a beam voltage, V₀, in the range of 10–30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V₀=1–3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V₀ to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed.     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

Evaluation of demineralized dentin contraction by stereo measurements using environmental and conventional scanning electron microscopy

Numerous investigations of etched human dentin are performed using scanning electron microscopy (SEM). Usually specimens are fractured and cross sections of etched layers with underlying unaffected dentin are observed. Results from this study showed that the edge of the etched layer contracted and became curved after fracture of wet specimens and that tensile stresses were developed in this layer by acid etching. The degree of contraction was determined utilizing profiles of the specimen edges obtained with the help of stereo measurements. Fixation in glutaraldehyde decreased the contraction in wet specimens prepared for environmental scanning electron microscopy (ESEM). Fixation also decreased shrinkage of the demineralized layer due to gradual desiccation in the ESEM during observation. For conventional SEM, the contraction was minimized if specimens of etched and fixed dentin were fractured in the dry condition after dehydration.     

Environmental scanning electron microscopy studies of diffusion mechanism of boron particle combustion

This investigation was performed to resolve long-term contradicting theories regarding the mechanisms which govern the species diffusion across the liquid B₂O₃ layer covering a single boron particle during the combustion of boron. An environmental scanning electron microscope (ElectroScan E-3) was used to observe the liquefaction characteristics of the boron oxide layer and to examine boron dissolution and species diffusion processes in real time. Using a hot stage, crystalline boron particles were heated from 25 to 950°C in O₂, H₂O, or Ar environments. Pure B₂O₃ particles were also heated in an O₂ environment and examined. In situ observations showed that the diffusion of dissolved boron into molten B₂O₃₍₁₎ is much more dominant at elevated temperatures than the diffusion of gaseous O₂ through the B₂O₃₍₁₎ layer. Dissolution of solid boron into the boron oxide layer caused the liquefaction of boron particles at relatively low temperatures (940°C). The chemical composition of liquid boron oxide, coated on the surface of boron particles, was identified as a polymeric vitreous (BO)_n complex through the reaction between dissolved boron and molten B₂O₃₍₁₎.     

Structure-preserving fixation allows scanning electron microscopy to reveal biofilm microstructure and interactions with immune cells

Pseudomonas aeruginosa is a pathogen that forms robust biofilms which are commonly associated with chronic infections and cannot be successfully cleared by the immune system. Neutrophils, the most common white blood cells, target infections with pathogen-killing mechanisms that are rendered largely ineffective by the protective physicochemical structure of a biofilm. Visualisation of the complex interactions between immune cells and biofilms will advance understanding of how biofilms evade the immune system and could aid in developing treatment methods that promote immune clearance with minimal harm to the host. Scanning electron microscopy (SEM) distinguishes itself as a powerful, high-resolution tool for obtaining strikingly clear and detailed topographical images. However, taking full advantage of SEM's potential for high-resolution imaging requires that the fixation process simultaneously preserve both intricate biofilm architecture and the morphologies and structural signatures characterising neutrophils responses at an infection site. Standard aldehyde-based fixation techniques result in significant loss of biofilm matrix material and morphologies of responding immune cells, thereby obscuring the details of immune interactions with the biofilm matrix. Here we show an improved fixation technique using the cationic dye alcian blue to preserve and visualise neutrophil interactions with the three-dimensional architecture of P. aeruginosa biofilms. We also demonstrate that this technique better preserves structures of biofilms grown from two other bacterial species, Klebsiella pneumoniae and Burkholderia thailandensis.     

Imaging of extraradicular biofilm using combined scanning electron microscopy and stereomicroscopy

Microorganisms are able to survive and induce persistent infection in extraradicular areas. The objective of this study was to evaluate the relationship between extraradicular biofilm and persistent periapical periodontitis. Thirty‐five apical samples with different stages of pulp and periapical pathology (vital pulp, pulp necrosis without radiographically visible periapical lesions, chronic periapical periodontitis that had not received root canal therapy and persistent periapical periodontitis) were initially evaluated using scanning electron microscopy. The same samples were then processed using Brown and Brenn‐modified Gram staining. We detected extraradicular biofilm in all samples with persistent periapical periodontitis and in three samples with chronic periapical periodontitis. The extraradicular bacteria predominantly had rod and filament morphology and were surrounded by varying amounts of amorphous extracellular material. The surfaces outside the root of the apical samples with vital pulp and pulp necrosis were covered by fibers, and no extraradicular microorganisms were present, which suggests that extraradicular biofilm is closely associated with failed endodontic treatments, thus resulting in persistent infection. Microsc. Res. Tech. 76:979–983, 2013. © 2013 Wiley Periodicals, Inc.