Specialised “wet-SEM” or “wet-TEM” specimen holders have also been developed for imaging of fully hydrated samples, but they require highly specialised equipment and work in scanning-transmission (STEM) mode, so are not directly comparable with SEM. Cryo-TEM is ideal for the investigation of macromolecular structures including viruses, however cryo-TEM gives a relatively small field of view, requires a high concentration of virus (~1 mg/ml) and most bacteria are too thick for high resolution TEM imaging in a layer of ice, which can obscure detail 16, 17, 18. This requires that specimens be maintained at temperatures below ~ −150 ☌ to remain in an amorphous state and avoid ice crystal damage. Cryo-TEM, a more advanced variant of this technique, can also be used to investigate frozen-vitrified samples in the TEM. Prior to SEM observation these frozen hydrated samples can either be cryo-sectioned, or mounted whole on a cryo-stage: in which case ion beam milling can also be used to investigate interior structure 13, 14. Flash freezing or high pressure freezing is often used to reduce ice crystal formation in biological specimens. Critical point drying permitted conductive coating of biological specimens, giving reduced charging as well as improvements in contrast, but the specimens suffered from cracking artifacts and shrinkage of up to 50%, while freeze-drying frequently causes distortion and damage due to ice crystal formation 15. Alternatively, methods to image specimens in the hydrated state have been used, employing “wet-SEM” 7, 9, 10, 11, environmental SEM 12, or cryo-techniques 13, 14. Previously, a variety of methods have been developed to dehydrate specimens prior to SEM observation, using solvents, sometimes in conjunction with critical point drying, or by freeze drying. During SEM observation drying is a problem and usually causes collapse, shrinkage and distortion of the specimen, even after preservation by chemical fixation. Both of these factors compromise microscope performance and can reduce contrast and resolution. If a wet specimen is placed in the microscope, operation under high vacuum conditions tends to dry the specimen out quickly. Secondly, biological specimens have traditionally needed to be dehydrated for the best imaging performance in the SEM. Firstly, in order to get adequate contrast and to reduce charging for small organic particles such as bacteria and viruses at magnifications greater than 1000 x, a conducting surface is needed. Two main problems occur with obtaining high resolution SEM images of microbes. These filters are suitable for surface observation of viruses and bacteria by SEM 7. Nowadays extremely high quality polycarbonate filters are available: the optimum pore size can be selected to collect any virus or bacterial species (the pores can be as small as 10 nm, less than the smallest viruses). The scanning electron microscope (SEM) can also be useful to reveal morphological features of isolated organisms as well as for diagnosis, but difficulty with specimen preparation methods have in the past limited the use of SEM for routine microbiology 7, 8. Electron microscopy is thus an ideal “catch all” method giving an “open view” for situations where a novel or emerging pathogen is being investigated where there is no a priori knowledge of the type of agent present 6. Moreover, electron microscopy is useful for identifying the type of microbe present, often to genus, allowing the selection of more specific tests (for example primers or specific antibodies) to fully identify the agents present. The recent development of filtration techniques show that both TEM and SEM identification of viruses can be carried out with as little as 5000 total particles per sample 7. By comparison, the level of detection of viruses using culture or nucleic acid testing usually ranges between 1 and 50 particles per assay 6. The detection of agents such as poxviruses or polyoma viruses in patient specimens usually requires a minimum concentration of between 10 5 to 10 6 particles/ml for TEM 4, 5. As a result, electron microscopy has historically suffered from low test sensitivity for many types of microbiological investigations. Thus microbes need to be grown to a high tire and/or concentrated by centrifugation, which is often not possible with patient specimens or agents that are not culturable. However, negative-stain TEM requires an adequate concentration of bacterial cells or virus particles, since these are adsorbed to a thin support film. ![]() Traditionally, negative staining for transmission electron microscopy (TEM) has been the “gold standard” for imaging microbial samples, for example in diagnostic virology 3. It is still an important technique that can help to diagnose pathogens and in testing to identify microorganisms 2. In early studies, electron microscopy was pivotal in helping to identify the causative agents of infectious diseases 1.
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