Diagnostic electron microscopy (EM) can contribute decisively to the laboratory diagnosis of infectious diseases and will be of particular importance if a rapid diagnosis is required, as in a potential bioterror scenario or if emerging infectious agents are to be characterized (O’Toole, 1999, Hazelton and Gelderblom, 2003, Madeley, 2003, Miller, 2003, Ng et al., 2003, Goldsmith et al., 2004, Morens et al., 2004). Likewise EM should be applied in critical clinical setting of controversial or urgent pathological cases (Kamondi et al., 2000, Nordhausen and Barr, 2001, Turbat-Herrera et al., 2004). In many instances, in infectious diseases a rapid and accurate diagnosis is achieved after negative staining of virus or bacterial suspensions (Madeley and Field, 1988, Biel and Madeley, 2001, Gelderblom, 2001, Gelderblom, 2003, Gentile and Gelderblom, 2005; Robert Koch Institute http://www.rki.de). However, in other cases, if infected cell cultures or tissues (e.g., biopsies taken to complement material for the negative staining approach) have to be examined, the need for rapid embedding methods enabling a same-day diagnosis from ultrathin sections is evident. In the past a number of simplified tissue embedding procedures using reduced dehydration times and increased polymerization temperatures were proposed (Bencosme and Tsutsumi, 1970, Johannessen, 1973, Doane et al., 1974, Rowden and Lewis, 1974, Miller, 1982, Bozzola and Russell, 1992).

Rapid diagnostic EM could probably benefit from the microwave employment, a technology that has been successfully applied in organic chemistry (De la Hoz et al., 2005), industrial food processing (Vaid and Bishop, 1998), sterilization of hospital waste (Celandroni et al., 2004), and also in the clinical laboratory. Here a great number of microwave accelerated procedures for light microscopy and EM tissue processing, staining reactions, and immunolabelling was developed (Mayers, 1970, Leong et al., 1985, Login and Dvorak, 1988, Login and Dvorak, 1994, Kok and Boon, 1990, Kok and Boon, 2003, Shi et al., 1991, Leong and Sormunen, 1998, Cavusoglu et al., 2001, Giberson and Demaree, 2001, Giberson et al., 2003, Leria et al., 2004, Munoz et al., 2004), all of them resulting in a significant time reduction. The effect of microwave irradiation on polar substances is mainly well understood. It is attributed to dielectric heating, also called “thermal effect” (Leonard and Shepardson, 1994, Giberson and Demaree, 1995, Kok and Boon, 2003) causing a temperature rise in the whole sample (“internal heating”; in contrast to conventional heating which starts at the specimen surface). The existence of an additional “non-thermal” effect of microwave energy (Marani and Feirabend, 1993, De la Hoz et al., 2005), which may be particularly effective in biological hydrated material in influencing the fixation of cells (Ruijgrok et al., 1993, Leria et al., 2004, Wendt et al., 2004), is still unproven.

A number of efforts has been undertaken to standardize the microwave-assisted procedures (Login, 1998). Different microwave bench “ovens” designed for the laboratory requirements are now commercially available. Assuming the rise in internal temperature as the primary mechanism responsible for the enhanced diffusion of reagents and protein cross-linking during fixation, microwave device manufacturers continually add features into their equipment to provide the end user with more control over the irradiated reaction process.

We report here on the application of a microwave device (REM, Milestone, Sorisole, Italy) recently developed for EM rapid tissue processing equipped with a customized software and a sample basket handling system compatible to our routinely used tissue processor (LYNX, Leica, Bensheim, Germany). Using this system, various specimens representing critical infectious or potential bioterrorism agents and liver biopsies which substitute pathological samples were processed in parallel following both a conventional and a microwave-assisted protocol. Ultrathin sections were evaluated, images were documented digitally and the results of both techniques compared.

A significant processing time reduction was realized between the conventional (3 days) and microwave-assisted protocol (4 h 25 min or 3 h 20 min, Table 1). As the solution exchange in the microwave “oven” is not automated, it had to be performed manually as well as the transfer of the samples into the BEEM-capsules for polymerization (including probe identity tags mounting), which required additional time depending on the number of samples processed in parallel (approximately 1 h).

The microwave processed resin blocks were polymerized uniformly and revealed good sectioning properties for semi- and ultrathin sections. The sections examined in the EM were stable in the electron beam. The images were crisp displaying a high and uniform contrast.

After microwave-assisted processing the fine structure of virus-infected cells and infectious agents was well preserved (Fig. 4, Fig. 5, Fig. 6, Fig. 7, Figs. 8 and 9, Fig. 10, Fig. 11 ). The SARS-coronaviruses can be clearly recognized in the dilated perinuclear space and RER-cisternae, also the microtubules of the cytoskeleton and mitochondria of the host cells are visible in detail (Fig. 4b). In cells infected with the elephant Orthopox virus artificial extraction of the core material was much less common (Fig. 5). This is a phenomenon often experienced in conventional embedding preparations. In the apathogenic B. anthracis and E. coli culture similar good preservation of the vegetative organisms as well as of mature Anthrax spores were achieved in both conventional LYNX and microwave-assisted embedding (Fig. 6, Fig. 7); each mature Anthrax spore displayed a clearly delineated endo- and exosporium covered with tiny surface microfilaments (Fig. 6b and d).Fig. 4

SARS-coronaviruses produced in Vero. 6 cell culture, conventional fixation (2.5% GA): (a), (b) REM-microwave embedding; (c), (d) LYNX-embedding; (e), (f) external embedding (Robert Koch Institute, Berlin). Original magnification: (a), (c), (e) 1250×; (b), (d), (f) 10,000×.

Short turnaround times as well as reliable good section and image quality are important criteria when rapid embedding protocols are considered. Comparing the samples processed according to a microwave-assisted versus a conventional protocol, we obtained very satisfying results with the microwave bench “oven”. The experienced reduction in sample processing time from days to hours with no compromised ultrastructural preservation of selected “difficult” specimens is in agreement with other workers using this technology. Microwave processing time of 3–4 h (Nordhausen and Barr, 2001, Gerrity and Forbes, 2003, Laboux et al., 2004) and short as 2 h (Giberson et al., 2003) were reported from EM laboratories with similar sample spectrum and workload as those of the authors.

The microwave-assisted processed samples were comparable or superior in section and image quality to the conventionally processed samples and compliant to published standards (Morioka et al., 1992, Miller, 2003, Peiris et al., 2003, Buchanan, 2004, Celandroni et al., 2004, Franzen et al., 2005, Larsson, 2005). The completely microwave-assisted processed parenchymal tissue of liver, used in our comparative approach as a substitute for other pathological solid tissues, revealed good results as shown in Fig. 12 in samples not exceeding 1-mm edge length. In the centre of the block where larger samples had been taken, there was a loss of ultrastructural preservation, and artefacts were seen such as have been described previously by Wild et al. (1989).

This phenomenon reflects the continuing discussion about the probably “dichotomous” nature of the non-ionizing microwave radiation in respect to the “heat” and the “non-thermal” or direct microwave effect (Galvez et al., 2004). Despite the complex aspects influencing the aldehyde fixation process of tissues (Fox et al., 1985, Leong and Sormunen, 1998, Izumi et al., 2000, Werner et al., 2000, Giberson and Elliott, 2001), recent observations tend to support the existence of the direct mechanism of microwave energy action (Kok and Boon, 2003, Leria et al., 2004, Wendt et al., 2004, De la Hoz et al., 2005, Gaber et al., 2005). This needs to be considered in the microwave-assisted tissue processing because also our results suggest, that an “overheating” inside the solid tissue samples can adversely affect tissue ultrastructure. Therefore, further development work should include efforts to reduce the critical “thermal” effect and increase direct “non-thermal” microwave action during tissue fixation (Galvez et al., 2004, Leria et al., 2004).

The REM Milestone microwave device is run with the sample baskets that are used also in the widely applied automated LYNX-processor for routine conventional tissue embedding. We observed that this shared equipment is a great benefit for the EM laboratory, e.g., a sample received late afternoon can be rapidly fixed in the microwave (e.g., 10–20 min in place of at least 2 h at room temperature) and added (without basket change) to other tissues which are to be processed automatically in the LYNX overnight anyway, and if urgent, rapidly polymerized in the microwave in 1 h 45 min the next morning (instead of 48 h). This option generates more flexibility of the laboratory workflow in handling clinical urgent samples and improves the economic employment of the available manpower. Thus, a “same-day” electron microscopy diagnosis for cells and tissues becomes reality.

The results obtained with the shortened protocol appear especially promising. As judged from sectioning properties and section quality, the samples were uniformly polymerized, i.e., comparable to the results with the longer microwave protocol. The observed denser appearance of cell cytoplasm could be explained by less extraction of cell material during the shorter course of the complete procedure. Therefore, in view of the acceptable results with the approximately 3.5 h protocol, attempts to cut down further the preparation schedule appear worthwhile.

Clearly, urgent samples reaching the diagnostic laboratory at unpredictable daytime need to be examined immediately in potential bioterrorism scenario or in case of an emerging agent disease (Lane et al., 2001, Morens et al., 2004). The microwave-assisted sample preparation enables EM to evaluate also thin sections for rapid diagnosis. Basing on our experience, the subsequent section ultramicrotomy, staining and examination in the EM can be completed in about 2 h. In consequence, using the potential of accelerated section preparation, EM-diagnosis from biopsy material can be rendered on the same workday. Thus, both negative staining of small particulate suspensions – from the smallest viruses up to bacteria, spores and parasites – as well as tissues and diagnostic cell cultures can be scrutinized by the “open view” of EM (Hazelton and Gelderblom, 2003) as part of the “frontline” diagnostic methods guiding further measures, e.g., containment and therapeutic activities. In combination with digital image acquisition and telemicroscopy networking tools (Schroeder et al., 2001) crucial or interesting findings could be consulted or shared worldwide in very short time.