Detection of Viruses by Electron Microscopy

1. Detection of Viruses byElectron Microscopy Lab 2

2. Electron microscopy • An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. • Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 0.2 nm resolution and magnifications of up to 2,000,000x, whereas ordinary light microscopes are limited by diffraction to about 200nm resolution and useful magnifications below 2000x. Transmission EM

3. Types of electron microscopes • Transmission electron microscope (TEM) • Scanning electron microscope (SEM) • Reflection electron microscope (REM) • Scanning transmission electron microscope (STEM) • Low-voltage electron microscope (LVEM)

4. Transmission electron microscope (TEM) • Electrons are emitted by an electron gun. The electron beam is accelerated by an anode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. • When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope.

5. TEM

6. Scanning electron microscope (SEM) • The SEM produces images by probing the specimen with a focused electron beam that is scanned across a rectangular area of the specimen. • Generally, the image resolution of an SEM is about an order of magnitude poorer than that of a TEM. However, because the SEM image relies on surface processes rather than transmission, it is able to image bulk samples up to many centimetres in size and has a great depth of field, and so can produce images that are good representations of the three-dimensional shape of the sample.

7. Virus visualisation • Perhaps the most obvious method of virus identification is direct visualisation of the virus itself. • The morphology of most viruses is sufficiently characteristic to assign an unknown virus to the correct family and, in the context of the particular case, the method may provide an immediate definitive diagnosis. • Non-cultivable viruses may also be detectable by electron microscopy.

8. Sample preparation • Chemical fixation • Negative stain: suspensions containing fine biological material are briefly mixed with a dilute solution of an electron-opaque solution such as ammonium molybdate, uranyl acetate (or formate), or phosphotungstic acid. This mixture is applied to a suitably-coated EM grid then allowed to dry.

9. Disadvantages of EM • Low sensitivity is the biggest limitation of electron microscopy as a diagnostic tool. Quite a bit of time is required (15 minutes or more) for a skilled microscopist, using a very expensive machine, to scan the grid adequately and detect viruses when the specimen contains fewer than 107 virions per milliliter. • Such levels are often surpassed in faeces and vesicle fluid, but not in respiratory mucus. Faeces are first clarified by low-speed centrifugation; the supernatant is then subjected to ultracentrifugation, to deposit the virions.

10. Identification of Viruses byImmunoelectron Microscopy • Definitive identification of virions may be achieved by adding specific antibody to the specimen before depositing the virus-antibody complexes onto the electron microscopic grid by centrifugation. • Negative staining reveals the virions as aggregates.

11. Summary • Advantages • useful when you don’t know what you’re looking for • useful for confirming identification • Disadvantages • requires specialised equipment and technicians • needs a lot of virus present • virus needs to be present at time of clinical signs • expensive • time consuming

12. Electron microscopy: Transmission EM Cat pox virus (EM: negative staining) Coronavirus (EM: negative staining)

13. Scanning EM