Abstract
Visualization of virus particles and morphological features depends on the resolution of microscopes. Transmission electron microscopy (TEM) is the starting point for obtaining the best resolution of images. Two different techniques are available and described in this paper. Firstly, negative staining of viral suspensions provides detailed information of virus particles' structure. It is a technique that can be quickly performed and is able to accommodate the highest magnifications of virus particles. Secondly, ultra-thin sections of virus-infected tissues or cell cultures, combined with a positive staining technique can provide information regarding the localization of viruses inside or around cells. These two complementary techniques for investigating the structure of a virus and its parasitic life cycle are presented in this paper.
Keywords
- transmission electron microscopy
- negative staining
- positive staining
1. Introduction
High resolution microscopes are essential for visualizing virus particles. A transmission electron microscope (TEM) fulfils this requirement and is widely used by scientists. Unknown agents of diseases may be quickly detected by using such a microscope. A recent review on the value of electron microscopy and virus structures was presented by Zhang et al. [1].
Shortly prior to the recent recognition of mimiviruses [2], orthopoxviruses had been one of the largest virus particles, in addition to the thin and very long filoviruses. Groups of orthopoxvirus particles can be detected when inside infected cells using the highest magnification available in light microscopy. Nevertheless, an isolated virus particle of ca. 250 nm diameter remains below the resolution of standard light microscopes. With the advent of TEMs and high magnifications of over 100 000x, the structural details of virus particles could then begin to be recognized.
The first demonstration of a viral particle using a TEM was in the form of a member of the orthopoxvirus genus [3]. Following the introduction of the negative staining technique in the 1960s [4], a variety of viruses has been discovered and classified based on morphological characteristics such as symmetry, the presence or absence of the envelope or spikes and the size of these projections [5,6,7,8]. A large number of viruses of medical importance that had formerly never been described such as adenovirus, enterovirus, orthomyxovirus, reovirus and paramyxovirus were identified by TEM after isolation in cell cultures inoculated with clinical specimens [9]. Other viruses like hepatitis B and hepatitis A were detected directly by TEM in samples such as plasma and faeces, following the failure of attempting to try and isolate them in cell cultures. The initial classification of many agents was therefore based on a combination of morphology and genome structure.
Currently, more than 30 000 different viruses comprising 56 families have been identified using TEM and humans have been found to play host to 21 of the 26 families specific for vertebrates [10]. The development of other techniques, e.g., immunofluorescence, enzyme-linked immunosorbent assays (ELISA) and biological molecular methods such as polymerase chain reaction (PCR) have progressively reduced the importance of TEM in virus diagnosis and microbiology. However, compared with other diagnostic methods, TEM still benefits from its rapidity and “open view”, i.e., the capability of detecting all pathogens present in a clinical specimen [11]. Therefore, TEM should be utilized as a frontline method in infectious disease emergencies and/or in suspected cases of bioterrorism [12]. Electron microscope studies were critical in identifying the aetiologic agent of severe acute respiratory syndrome (SARS), a coronavirus, during its 2002/2003 global outbreak [13].
Several methods for specimen preparation have been developed. These can be summarized into two procedures: the negative staining of vesicular content and viral suspensions, and the ultra-thin sectioning of infected tissues and cells. Both techniques have been carried out with complementary results.
2. Negative staining technique
The classic processing of biological specimens observed in a TEM needs fixation, dehydration, sectioning and a selective “staining” of cell and tissue structures. “Staining”, a means of receiving coloured images, cannot be effectively used in conjunction with an electron microscope. Instead, the enhancement of structures for TEM observation is effected, usually by impregnation with heavy metal salts of plumb, tungstenium and uranium.
Some biological elements are very small and as a result, sectioning reveals aspects of its content but not about its global surface structure. The result is a bi-dimensional image. Nevertheless, when observed as a whole using the negative staining technique, these elements reveal a tri-dimensional image.
To prepare small biological specimens such as bacteria, viruses, phages, micoplasma, filaments and cell membranes, or even nucleic acids and protein filaments for TEM observation, the special technique of “negative contrast” was developed [4]. Instead of applying a more or less strong positive contrast of structures (“staining”), in this instance, contrast is not applied to the object but to its environment, using heavy metal salts. The electron beam can cross biological material easier than the surrounding space. The result resembles an inverted traditional TEM image (Figures 2, 4-7).The standard staining solution used today is an aqueous 1% phosphotungstic acid with pH 7.2.
Essentially, a fixed or unfixed drop of viral suspension is applied onto a formvar- or collodium-covered electron microscope grid for a few seconds; the liquid is absorbed by a filter paper, then a drop of the staining solution is applied and few seconds later also absorbed (Figure 1). After drying, the specimen is ready to be introduced into the electron beam.
This unbelievably simple technique, at the outset rejected by the scientific community, eventually became a revolutionary approach for studying primarily viral morphology as emphasized by viral diagnostics [14]. Extremely detailed images were obtained and published, revealing substructures and macromolecules as viral antibodies and virus particle spines [5, 9, 15, 16, 17, 18, 19, 20].
In order to gain additional information about small structured elements, several technical steps in sample processing were developed such as immune-electron microscopy (Figure 5), solid-phase-electron microscopy, ammonium-sulphate precipitation, gradient fraction contrasting and particle concentration by diffusion in an agarose layer [21].
Contrasting solutions show a large spectrum of possibilities. Selection must be in accordance with the pH and electrical charge of the sample, of the contrasting solution and of the EM grid or support [19, 22]. Resulting precipitates and a lack of spreading of the sample are the most common inconveniences. For better spreading and adsorption of virus particles onto a formvar-carbon coated grid, polylysine (poly-L-lysine) is currently being used in our laboratory (Figures 6 to 7).
An overview of the negative staining technique development and application was presented by Biel & Gelderblom [6], Harris et al. [23] and more recently, by Schramlová et al. [24]. Benefits or deception are always surprising factors when the electron beam reveals a TEM image inside a dark room.
3. Positive staining technique
The positive staining technique has been used since the late 50s and the early 60s for enhancing the contrast of biological samples (tissues and cell structures, viruses, etc.). Using this technique, as well as negative staining, the samples are incubated in heavy metal salt solutions that react with cellular structures. Uranyl acetate [25] and lead citrate [26] are the most commonly used salts today. Grids containing ultra-thin sections of a sample are incubated for 15 minutes in uranyl acetate; this procedure should be performed in an environment protected from light. Following on, the grids are washed in distilled water and incubated in lead citrate at four to five minutes in an environment free of CO2. NaOH tablets are used to keep the environment free from reacting with CO2 (Figure 8). At the end of the procedure the grids are washed in distilled water, air dried and observed with a TEM [27, 28].
3.1. Specimen preparation
4. Advantages and disadvantages of the TEM
Virus diagnosis via TEM, when compared to other techniques such as immunofluorescence, PCR and ELISA have both advantages and disadvantages.
Acknowledgments
Financial support: research fellowship of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), process numbers 301525/2009-9 and 304067/2014-18 (second author).
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