To provide contrast in the electron microscope
Visualization of individual virus particles
Most biological materials show little contrast with their surroundings unless they are stained. In the case of light microscopy, contrast can be enhanced by using coloured stains which selectively absorb certain wavelengths. The electrons in the electron microscope are absorbed very little by biological material and contrast is obtained mainly by electron scattering.
To heighten the contrast between viruses and the background, use is made of electron-dense "stains". These are usually compounds of heavy metals of high atomic number, that serve to scatter the electrons from regions covered with the stain. If virus particles are coated with stain (positive staining), fine detail may be obscured. Negative staining overcomes this problem by staining the background and leaving the virus relatively untouched. The negative stain is moulded round the virus particle, outlining its structure, and is also able to penetrate between small surface projections and to delineate them. If there are cavities within the virus particle that are accessible to the stain, these will be revealed and some of the internal structure of the virus may be disclosed.
For negative staining, use is made of transmission electron microscopy, and only electrons which pass through the specimen are involved in the formation of the final image.
When particles are covered with stain, components of both top and bottom surfaces of a three-dimensional structure are contained in the two-dimensional image. This superposition can make it difficult to distinguish fine structures which would ordinarily be well within the resolution of the electron microscope.
One-sided staining provides more accurate information about the organization of capsomers, etc., although these particles are less well supported and tend to "collapse", resulting in apparent increase in size.
When two surfaces of regularly repeating units (such as the surface of some virus particles) are superimposed slightly out of register, moire pattern artefacts may result which could lead to false interpretation of particle ultrastructure.
Below is an example of rotaviruses stained from below, and from both sides (top and bottom)
Rotaviruses have a lattice-like arrangement of capsomers (left), and the large ring shapes (right) are artefacts produced by moire effects.
Superposition artefacts are seen in this semi-crystalline array (left) of an isometric virus from the insect Nudaurelia.
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