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.     

High-pressure scanning electron microscopy of insulating materials: A new approach

A new SEM technique for imaging uncoated non-conducting specimens at high beam voltages is described which employs a high-pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation. A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.     

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.     

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.     

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.     

A technique for comparative light and scanning electron microscopy of the same deparaffinized microtomed section*

Evaluation of various types of clear plastic support material for light and scanning electron microscopy of the same section was conducted. GelBond was found to be the most satisfactory among seven different types of plastic materials tested. It offers all of the advantages of glass slides for light microscopy while being thin and flexible enough to cut and sputter coat for SEM.     

Environmental scanning electron microscopy investigations of biodeterioration

Case studies will be presented in which environmental scanning electron microscopy (ESEM) has been used to provide unique insight into the role of microorganisms in deterioration processes. ESEM is an excellent tool for demonstrating spatial relationships between microorganisms and substrata because hydrated, nonconducting samples can be viewed with a minimum of manipulation. Copper and iron-rich deposits associated with bacteria were detected within corrosion layers on copper and steel surfaces, respectively. Fungal mycelia growing on wooden storage spools were shown to penetrate protective grease on carbon steel wire rope in contact with the spool and to cause localized corrosion. Large numbers of marine bacteria were documented within paint blisters and disbonded regions of fiber-reinforced polymeric composites. In both cases, it appears that microbial gas production resulted in mechanical damage to the substrata.     

A portable cryo-storage system for low-temperature scanning electron microscopy,suitable for international transport of cryo-specimens

A cryo-specimen storage system for low-temperature scanning electron microscopy (LTSEM) specimens is described, which: liberates multi-specimen experiments from sampling restrictions imposed by the rate at which LTSEM specimens can be examined in the SEM; provides security against experiment loss resulting from breakdown of the SEM or cryo-system; enables collection of specimens in the field or in laboratories remote from the SEM laboratory; and facilitates international air transport of LTSEM specimens. The components of the system, which has a capacity of 98 stub-mounted specimens, are readily made in a laboratory workshop. The details of the design may be altered to suit particular specimen types or experimental approaches.     

Application of channel electron multipliers in an electron detector for low‐voltage scanning electron microscopy

An electron detector containing channel electron multipliers was built and tested in the range of low‐voltage scanning electron microscopy as a detector of topographic contrast. The detector can detect backscattered electrons or the sum of backscattered electrons and secondary electrons, with different amount of secondary electrons. As a backscattered electron detector it collects backscattered electrons emitted in a specific range of take‐off angles and in a large range of azimuth angles enabling to obtain large solid collection angle and high collection efficiency. Two arrangements with different channel electron multipliers were studied theoretically with the use of the Monte Carlo method and one of them was built and tested experimentally. To shorten breaks in operation, a vacuum box preventing channel electron multipliers from an exposure to air during specimen exchanges was built and placed in the microscope chamber. The box is opened during microscope observations and is moved to the side of the scanning electron microscope chamber and closed during air admission and evacuation cycles enabling storing channel electron multipliers under vacuum for the whole time. Experimental tests of the detector included assessment of the type of detected electrons (secondary or backscattered), checking the tilt contrast, imaging the spatial collection efficiency, measuring the noise coefficient and recording images of different specimens.     

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.     

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.     

A robust focusing and astigmatism correction method for the scanning electron microscope—Part III: An improved technique

As described in a previous work, a new technique has been developed to perform automatic focusing and astigmatism correction on any general scanning electron microscope (SEM) sample ranging from gold-on-carbon to integrated circuit (IC) tracks. In this work, various improvements made to this technique are reported. They include the implementation of direct control of the SEM and the development of three new algorithms, namely the adaptive fast Fourier transform (FFT) algorithm, the coarse focusing algorithm, and the fine focusing algorithm. Direct control reduces the communication time with the SEM while the three new algorithms are integrated with the existing technique to make it even more robust to noise and to extend it to correct images with any degree of defocus and astigmatism. The enhanced focusing and astigmatism correction algorithm is able to perform the correction in a shorter time while maintaining the accuracy of the original algorithm even under noisy conditions.     

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.     

Application of the technique of environmental scanning electron microscopy to the paper industry

The environmental scanning electron microscope was used in conjunction with a heating stage to study in situ the polymerization process in the manufacturing of filter papers for the automotive industry. The images obtained from the video recording were converted into TIFF format with a computer program and were then analyzed using an image analyzer to assess the percentage shrinkage during the cure process. These experiments yield valuable information that is impossible to obtain with other electron microscopy techniques, for example, determining whether the polymer envelops the fibers or not, or whether there is a greater or lesser degree of anchoring between fibers.     

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.     

Primary considerations for image enhancement in high-pressure scanning electron microscopy

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high-pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist-environment ambient-temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross-sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high-pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high-pressure SEM are considered in detail in the accompanying paper.     

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.     

A rapid and simple technique for correlating light microscopy,transmission and scanning electron microscopy of fixed tissues in Epon blocks

A simplified and standardized technique for close correlation between light microscopy (LM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) is described. Perfusion and immersion fixed tissue specimens were embedded in Epon 812 and cut for conventional LM and TEM. The Epon blocks with remaining tissue were thereafter treated with epoxy solvent (ethanol-NaOH solution) for partial epoxy resin removal only (dissolving rate approx 33μm/h). The blocks with partially blotted tissue specimens were then critically point dried and gold coated for SEM. This method, in an easy way, allows repeated observations with LM, TEM and SEM with preserved fine structure and exact correlation. Since the technique is so simple and there is no need for special equipment the method can easily be adopted in all laboratories with basic SEM standards.     

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.