- Viral diagnosis by direct or indirect visualization can be achieved by using light or electron microscopy and relies on the identification of viruses based on typical morphological characteristics.
- Noncultivable viruses may also be detectable by electron microscopy.
Light Microscopy
- Are seldom used to visualize viruses, as they lack sufficient magnifying or resolving power to do so.
- However, the largest known viruses can be seen with a light microscope.
- Examples of these very large viruses include Mimivirus, Pithovirus, Megavirus, and Pandoravirus, all of which infect amoebas
- Indirect evidence of viral infection can be detected by light microscopy
-inclusion bodies (composed of masses of virions), multinucleated cells, and syncytial cells - Also, Imaging of viruses by light microscopy whereby techniques such as Immunostaining or Immunofluorescence labelling can be employed so that proteins of interest of the virus, can be labelled with antibodies which are fluorescently tagged.
- Viral inclusions can be found in the nucleus or the cytoplasm of infected cells
- The location is characteristic of the responsible virus
- Principle: visualize an image by using a glass lens, and magnification is determined by, the lens’s ability to bend light and focus it on the specimen, which forms an image.
- Advantages: Light microscopy is cheap and easy to apply. It is possible to observe virally induced changes to infected cells even thought many viruses are too small.
- Limitations: Lack sufficient magnifying and resolving power. It is not suitable for intense visualization of smaller viruses.
Fluorescence Microscopy
- This refers to the microscopic technique that involves emission of light by a substance that has absorbed light or other electromagnetic radiation.
- It irradiates a specimen with high-energy excitation light and detects the weaker emitted fluorescence.
- A viral diagnostic assay called an immunofluorescence assay (IFA) uses tagged antibodies to detect viral proteins in infected cells.
- Another way to add a fluorescent tag to a protein is to use genetic engineering (cloning) where the Green Fluorescent Protein (GFP) gene is introduced into a cell and the protein product is visible when cells are exposed to UV light.
- Principle: The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different colour than the absorbed light).
- Advantages: Sensitive, specific and simple and fluorophore labelled molecules are very bright and readily distinguishable from
other background signals.
Limitations: Fluorophores lose their capacity to fluoresce when illuminated due to photobleaching. Cells are prone to phototoxicity, especially when a short wavelength is used. Addition of a relatively large protein tag can alter the trafficking’ localization or enzymatic activity of the protein of interest.
Electron Microscopy
- Electron microscopy (EM) has been used for many years for the rapid detection of viruses in clinical specimens.
- During the 1970s electron microscopy was the means to the discovery in feces of several new groups of previously non-cultivated viruses.
- The human rotaviruses, caliciviruses, astroviruses, hepatitis A virus, and previously unknown types of adenoviruses and coronaviruses were all initially identified in this way.
- This technique relies on the identification of viruses by their characteristic morphology.
- Principle: Electrons are such small particles that, like photons in light, they act as waves. A beam of electrons passes through the specimen, then through a series of lenses that magnify the image. The image results from a scattering of electrons by atoms in the
specimen.
Advantages: High resolution. High resolution microscopes are essential for visualizing virus particles.
Limitations: The virus must be present in sufficient quantity (approximately 105–106 particles/mL) in order to be detected. Fixed and processed samples are dead and samples are heavily damaged by electron beams. The most potent usefulness lies in detecting viruses in fecal contents; EM is not used widely for routine diagnosis because it is expensive, cumbersome, and insensitive. Newer rapid tests are available for most viruses that previously were diagnosed by EM. - Types of EM: There are two main types of electron microscopes (EM), the scanning EM (SEM), and the transmission EM (TEM).
- (a) SEM: The main parts to an SEM are: source of electrons, a column for them to travel with electromagnetic lenses, an electron detector, sample chamber, and a computer and display to view the images. A high energy beam of electrons is aimed at the sample. As they interact with the sample, secondary electrons and X-rays are produced. Those signals are collected by the detectors and an image is formed on the computer screen.
- (b)TEM: The TEM is a little different than the SEM. A tungsten filament is used to make an electron beam in a vacuum chamber, then, instead of bouncing off the surface of the sample, the beam passes through it. The sample is a very thin slice of material, less than 100nm. When the electrons pass through, they hit a phosphor screen, CCD or film and an image is made. TEM can have a resolution of 0.2nm, that is smaller than the size of most atoms. The TEM can show the structural arrangement of atoms in a sample material.
Two general procedures can be applied to virus detection by electron microscopy: negative and positive staining electron microscopy.
Negative staining electron microscopy
- Negative staining is a procedure, which embeds small biological particles adsorbed on an electron transparent sample support (EM grid) in a thin and amorphous film of heavy metal salts to reveal their structural details in the transmission electron microscope (TEM).
- A negative staining technique uses heavy metal salts of plumb, tungstenium and uranium to enhance the contrast between the background and the virion’s image.
- 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.
- In Negative staining, 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 .The standard staining solution used today is an aqueous 1% phosphotungstic acid with pH 7.2.
- Advantages: It is possible able to view the cells without risk of them being damaged or distorted as they might be in positive
- Disadvantages: Artifacts caused by drying, dehydration and flattering of specimens. Does not provide much information about cell size rather than the cell size, shape and arrangement.
Positive staining electron microscopy
- 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.).
- Positive staining stains the virus itself such that the virus is dark against a lighter background.
- This is the easiest staining to do. It is “quick and dirty”. It provides less detail of the viruses, but will allow to measure their dimensions. It is not the proper staining method if you want really pretty micrographs of the viruses.
- Using this technique, as well as negative staining, the samples are incubated in heavy metal salt solutions that react with cellular structures. Uranyl acetate and lead citrate 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. At the end of the procedure the grids are washed in distilled water, air dried and observed with a TEM.
Positive vs negative staining
