Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.     

Rapid diagnosis of malaria by fluorescence microscopy

The routine procedure for detection of blood stages of Plasmodium spp involves Giemsa staining of thin and thick blood smears. This procedure, although simple, is time consuming, and its interpretation is dependent upon the training and experience of the observer. New methods for malaria diagnosis still require considerable financial outlay for specialist equipment and re-training of staff In this article, Hiko Kawamoto and Peter Billingsley discuss an efficient method for the detection of malaria parasites using low-cost, paired filters adapted for standard light microscopes and acridine orange staining.     

Rapid diagnostic thin section electron microscopy of bacterial endospores

Emerging infectious diseases such as SARS and the bioterror attacks with anthrax spores that occurred after September 11th, 2001 have highlighted the need to be better prepared for the detection and management of infectious pathogens that threaten public health. Negative staining electron microscopy is one method used to screen environmental and clinical samples for relevant infectious pathogens. Unfortunately, bacterial endospores, like those of Bacillus anthracis, are difficult to identify using this method because of their density that prevents imaging of structural details. Thin section electron microscopy would be an alternative method but this usually requires a few days for preparation and diagnosis. In the present paper we describe the development of a rapid thin section protocol, using mainly Bacillus subtilis spores as a model, which allows an unequivocal diagnosis of endospores within 2 h. The protocol involves chemical fixation assisted by heat or microwaves, rapid dehydration, embedding in the low-viscosity resin LR White and chemically enhanced polymerization. Structural preservation of spores is comparable to preservation after standard Epon embedding. Immunolabeling experiments using B. atrophaeus spores and a specific antibody suggest that the protocol preserves significant antigenicity for on-section immunocytochemistry and therefore offers the possibility for the strain typing of spores using specific antibodies. Further experiments with vegetative bacteria, viruses and cell cultures indicate that the rapid thin section protocol not only preserves spores but also other biological structures. Because of its universality and speed the described protocol complements negative staining electron microscopy as a front line method for the morphology-based diagnosis of pathogens in environmental and clinical samples.     

Rapid polymerisation with microwave irradiation for transmission electron microscopy

Successful results of microwave polymerisation of different epoxy formulations have been reported in the literature. The present study was intended to shorten the time needed for polymerisation of epoxy resin by the use of a microwave technique. A standard double fixation and tissue processing was applied to samples of rat kidney tissue. Tissue samples from the control group were polymerised in a conventional oven at 60 degrees C for 48 h, while tissue from the experimental group was irradiated in a microwave oven, initially at 900 W for 10 min and then at 360 W for another 100 min. During this irradiation, the sealed BEEM capsules were submerged in a water bath, so that the temperature rise was uniform and constant. This resulted in a homogeneous and rapid polymerisation. The cutting properties of the blocks in both groups were similar and no noticeable difference in the quality of the sections was evident when evaluated with TEM. The results showed that the use of a microwave oven reduced the time needed for the polymerisation of Epon blocks without any loss in quality.     

Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis

Electron microscopy(EM) should be used in the front line for detection of agents in emergencies and bioterrorism,on accounts of its speed and accuracy.However,the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results.Therefore,the research on viral structure and morphology has become important in EM diagnostic practice.EM has several technological advantages,and should be a fundamental tool in clinical diagnosis of viruses,particularly when agents are unknown or unsuspected.In this article,we review the historical contribution of EM to virology,and its use in virus differentiation,localization of specific virus antigens,virus-cell interaction,and viral morphogenesis.It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy.Furthermore,avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.     

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

Rapid microscale procedure for visualizing intracellular plasmid DNA by electron microscopy

A rapid microscale procedure is described that releases plasmid DNA in situ from the bacterial cell and that allows selective observation of the plasmid bound to cellular components. The released plasmid DNA was adsorbed preferentially on mica in a divalent cation-free medium then processed for electron microscopy. The plasmid DNAs studied were pAO3 (1683 base pairs (bp²)), λdv021 (3505 bp), pTSO118 (4000 bp), pAO65 (4786 bp), ColE1 (6500 bp), and RSF2124 (11,400 bp). These DNAs were seen as supercoiled circles or as relaxed circles of corresponding length. Occasionally an internal loop of replicating DNA was present. One micrometer of measured length corresponded to 3100 bp.     

Rapid phenotypic analysis of uncoated Drosophila samples with low-vacuum scanning electron microscopy

Research projects featuring repetitive phenotypic analysis of insects, such as taxonomic studies, quantitative genetics, and mutant screens, could be greatly facilitated by a simpler approach to scanning electron microscopy (SEM). Here, we have applied low-vacuum SEM to wild type and mutant Drosophila and demonstrate that high quality ultrastructure data can be obtained quickly using minimal preparation. Adult flies, frozen live for storage, were mounted on aluminum stubs with carbon cement and directly imaged, with no chemical treatment or sputter coating. The key imaging parameters were identified and optimized, including chamber pressure, beam size, accelerating voltage, working distance and beam exposure. Different optimal conditions were found for eyes, wings, and bristles; in particular, surface features of bristles were obscured at higher accelerating voltages. The chief difficulties were charging, beam damage, and sample movement. We conclude that our optimized protocol is well suited to large-scale ultrastructural phenotypic analysis in insects.     

Plasmodium: high-voltage electron microscopy

Stereoscopy of thick sections (~1.0 μm) of biological material, Plasmodium gallinaceum and Plasmodium berghei, was investigated with a high-voltage electron microscope using an accelerating voltage of 650 kv. Not only could structural details of the malarial parasites be observed in the thick sections, but stereoscopic examination also revealed the physical relationships of the various subcellular organelles in the parasites. This study indicates that the high-voltage electron microscope is a useful tool for structural analysis of malarial parasites.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.