Preparation of the nematode-trapping fungus,Arthrobotrys oligospora,for scanning electron microscopy by freeze substitution

A freeze-substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at — 20°C and critical-point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three-dimensional arrangement.     

Low-temperature scanning electron microscopy in fungus-nematode interaction

A comparative study has been made of different preparation techniques used for the scanning electron microscope (SEM), with regard to their application to fungus-nematode interaction. The preparation of frozen-hydrated specimens of both healthy and Arthrobotrys-oli-gospora-infected second-stage larvae of the root-knot nematode (Meloidogyne sp.) is described, and the results are compared with those obtained by critical point-drying and freeze-drying. In all cases the frozen-hydrated specimens consistently showed the best preservation.     

A simple method for SEM examination of sectioned diatom frustules

We describe an innovative yet straightforward method to obtain high quality thin sections of diatom exoskeletons for observation by scanning electron microscopy (SEM). The use of this new technique allows for clear observations of some ultrastructural valve features, including the raphe, which are generally difficult to observe and describe accurately using transmission electron microscopy analysis of thin sections or SEM of randomly fractured diatom valves. In addition, because this method involves the complete removal of the organic content of the diatom cells, resulting in clean and mostly undisturbed skeletal thin cross-sections, even the intact valvar structures of weak girdle bands can be studied.     

The axonal transmission of Herpes simplex virus to epidermal cells: a novel use of the freeze substitution technique applied to explant cultures retained on cover slips

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a technical challenge. We developed a technique to study the axonal transport of the Herpes simplex virus from dorsal root ganglia sensory neurones to epidermal cells. Autologous explants of human foetal dorsal root ganglia and skin were cultured on plastic cover slips. Axon fascicles grew from the ganglia to the epidermal cells and the ganglia were inoculated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from paraformaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, and embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the resin block, which retained the tissue. The resin block was placed in fresh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block. After trimming, serial sections were easily obtained and successfully immunolabelled for viral proteins. This is a convenient technique for immunolabelling tissue grown on cover slips in which the preservation of the ultrastructural interactions between different cells is important. It should be adaptable to a number of cell-culture applications and has a number of advantages over other techniques.     

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.     

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.     

Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole

The intravacuolar network inside the parasitophorous vacuole of Toxoplasma gondii consists of an intricate system of membrane-limited tubules of uncertain role in parasite development. We propose that it is an important structural support to the maintenance of the parasites in the characteristic rosette arrangement of parasites inside the vacuole, rather than being associated with the nutrient acquisition from the host cell, as previously suggested. We based our assumptions on observations made by field emission scanning electron microscopy of an epithelial cell line (LLCMK2) infected at various time intervals. Scraping the surface of infected monolayers with Scotch tape exposed the inner organization of the parasitophorous vacuole. Ultrathin sections and freeze-fracture replicas of analogous samples were correlated with field emission observations and added new data on tubular membranes and general organization of the parasitophorous vacuole.     

Scanning electron microscope design for quantitative multicontrast

Conceptual designs of scanning electron microscopes (SEMs) using a time-of-flight electron spectrometer are presented. The procedure for making quantitative measurements with such SEMs is shown to be much simpler and versatile than using conventional SEMs. SEMs which use an electron time-of-flight spectrometer are able to operate as multicontrast analytical probes, capable of simultaneously quantifying surface topography, voltage, and material type. In addition, it is demonstrated that these SEMs can be designed to have high spatial resolution, good signal-to-noise characteristics, and to be of compact table-top size.