Appendix 1 – Transmission Electron Microscopy in Virology: Principles, Preparation Methods and Rapid Diagnosis Hans R. Gelderblom Formerly, well-equipped virology institutes possessed many different cell culture types, various ultracentrifuges and even an electron microscope. Tempi passati? Indeed! Meanwhile, molecular biological methods such as polymerase chain reac- tion, ELISA and chip technologies—all fast, highly sensitive detection systems— qualify the merits of electron microscopy within the spectrum of virological methods. Unlike in the material sciences, a significant decline in the use of electron microscopy occurred in life sciences owing to high acquisition costs and lack of experienced personnel. Another reason is the misconception that the use of electron microscopes is expensive and time consuming. However, this does not apply to most conventional methods. The cost of an electron-microscopic preparation, the cost of reagents, contrast and embedding media and the cost of carrier networks are low. Electron microscopy is fast, and for negative staining needs barely 15 min from the start of sample preparation to analysis. Another advantage is that a virtually unlimited number of different samples can be analysed—from nanoparticles to fetid diarrhoea samples. A.1 Principles of Electron Microscopy and Morphological Virus Diagnosis In transmission electron microscopy, accelerated, monochromatic electrons are used to irradiate the object to be imaged. This leads to interactions: the beam electrons are scattered differentially by atomic nuclei and electron shells, losing some of their energy. After magnification through a multistage lens system, a 1,000-fold higher resolution is obtained with a transmission electron microscope than with a light microscope (2 nm vs. 2 mm) owing to the much shorter wavelength of electrons in comparison with visible light. Hence, in contrast to light microscopy, transmission electron microscopy is capable of visualizing even the smallest viruses. Scanning electron microscopy depicts surfaces, but no internal structures. Even though confocal laser scanning light microscopy can localize individual viruses, the fluorescence signal does not image the particle. However, identification S. Modrow et al., Molecular Virology, DOI 10.1007/978-3-642-20718-1, 949 # Springer-Verlag Berlin Heidelberg 2013 950 H.R. Gelderblom of viruses and description of virus–cell interactions frequently require structural information in high resolution, as can be supplied only by electron microscopy. Electron microscopes are also used in the diagnosis of viral diseases: although a modern diagnostic laboratory allows high sample throughput, it requires a clinical diagnosis and pathogen-specific reagents. By contrast, electron microscopy “illu- minates,” after rapid and simple contrasting, and provides a high-resolution “open view” of the finest structures in a sample. Thereby, every pathogen is rendered visible also in the case of multiple infections or unknown pathogens, or agents that have never been considered by the clinician, and even genetically engineered organisms. Infectious agents exhibit constant morphological characteristics such as size, shape, subunits, perhaps an envelope, surface projections or protrusions and specific nature of the interaction with the host cell. These criteria help to assign the object in question to a specific pathogen family. Although the morphological diagnosis “viruses of the herpesvirus family” does not indicate the viral species, as a “family diagnosis” it gives the clinician or epidemiologist—along with the previous clinical history—a rapid perspective on the action required, e.g. antiviral therapy, quarantine or vaccination. If the pathogen family is ascertained, if neces- sary, even a “fine diagnosis” by molecular methods or by immune electron micros- copy can be significantly accelerated. Independence from pathogen-specific reagents is a great advantage for diagnosis of infections, epidemics and bioterrorism hazards, but also in the characterization of laboratory products and biological preparations, vaccines, therapeutic antibodies and in the frequently neglected internal quality assurance. A disadvantage of electron microscopy is that it has a very narrow field of view, owing to the very high magnification; therefore, high particle concentrations are required for detection (more than 105 particles per millilitre). Samples with particle concentrations below this threshold need to be effectively and rapidly enriched before use. Furthermore, electron microscopy does not allow high-throughput analyses. The examiner must be concerned only with one preparation for at least 20 min before it can classify it as “negative.” A.2 Transmission Electron Microscopy: Thin Preparations Are Required In preparations with a layer thickness of more than 80 nm, the imaging electrons are scattered several times; the consequences are loss of image sharpness and informa- tion. The necessary thinner specimens are obtained after embedding the samples in resin (Epon) in ultrathin sections or by negative staining of particle suspensions. Both methods result de facto in a resolution of 2 nm. Viruses are biological macromolecules which consist essentially of light atoms with low mass density. In the transmission electron microscope, they appear highly transparent and with low contrast. Detailed and contrast-rich images can be obtained by absorption and scattering of electrons only at high mass density of the objects, as can be created by negative or positive staining (Fig. A.1). Appendix 1 – Transmission Electron Microscopy in Virology 951 Fig. A.1 Preparation for transmission electron microscopy (TEM). Principle of ultrathin-section and negative-contrast TEM using the example of a herpesvirus. The upper half shows the ultrathin section of a specimen embedded in Epon. For this purpose, in vitro infected cells were fixed with glutaraldehyde, incubated with heavy metal salts, dehydrated and finally embedded in resin. The hardened samples were cut in an ultramicrotome into 50-nm-thick specimens, treated again with contrast medium and analysed by TEM. The following viral structures can be recognized from outside to inside: the viral envelope as a lipid bilayer with glycoprotein protrusions, a moderately contrasted tegument, the hexagonal viral capsid and deep inside the viral DNA complex. Heavy metal salts are also used as contrast media in negative staining (lower half) (see Fig. A.2). In this case, the short exposure leads only slightly to chemical bonds. The viral components are trans- parent, they appear bright, negatively contrasted against the dark, electron-dense contrast medium, and we can recognize the fragile envelope of a herpesvirus with its glycoprotein processes and inside the contrast-medium-filled capsid with capsomeres and a typical diameter of 100 nm Negative staining requires particle suspensions, as they can be directly prepared from swab samples from patients, cell culture supernatants, disrupted cells, pulver- ized tumours, urine, serum or stool samples, bacterial colonies and bioterrorism- suspected “powder.” In principle, any organic or inorganic material is suitable if it can be resuspended. Rough cell debris is removed by low-speed centrifugation. The sample is then stained (Fig. A.2). Usually, a copper grid (carrier network) is placed on a drop of the suspension. The grid carries a stable and very electron transparent plastic carrier film with regularly dispersed holes. After short 952 H.R. Gelderblom Fig. A.2 Negative staining procedure. Drops of the suspension to be examined, washer fluid and contrast medium are applied from left to right in a horizontal row on an inert surface. Grids (in the Petri dish) are placed on the sample using a pair of tweezers for 10 s or longer, are then washed with drops of double distilled water and are finally stained with contrast medium for 10 s. After excess contrast medium has been removed and subsequent air drying, the grids are well prepared for TEM. (From Gelderblom 2003) adsorption, the sample excess and interfering ions are removed with filter paper and the grid with the adsorbed material is placed for a few seconds on the contrast medium, which consists of a 0.5–4.0 % heavy metal salt solution with high mass density, such as phosphotungstic acid or uranyl acetate. Excess contrast medium is removed from the “contrasted” grid, briefly air-dried and is then prepared for electron-microscopic examination. The dried, electron-dense contrast medium coats the “transparent” structures on the carrier network and renders visible surface details with very high resolution—comparable to the X-ray contrast technique. However, it can also penetrate into labile samples, facilitating the representation of internal structures. Advantageous for a natural image is that viruses, like other unstable biostructures, are closely enveloped, and thus are stabilized by the contrast medium. Thin specimens make possible, apart from negative staining, also the more complex ultrathin-section technique. For this purpose, small blocks of solid tissues, cell culture conglomerates or ultracentrifuge sediments (edge length under 1 mm) are chemically fixed by aldehydes, and incubated with heavy metal salt solutions (osmium, uranium, lead). The heavy metal salts bind to different groups in the material and this results in high mass densities and high-contrast images even of Appendix 1 – Transmission Electron Microscopy in Virology 953 internal structures, which become visible later. After dehydration,