Preparation of sections for the Electron Microscope

How do we make sections for the electron microscope?

Tissue blocks are generally fixed in glutaraldehyde, (whereas light microscopy normally uses paraformaldehyde).

They are postfixed in osmic acid.

The tissue is then dehydrated, and transferred to propylene oxide, which will mix with the resinous embedding medium. This is hard so that thin sections can be cut (60 to 80nm thick). The sections need to be this thin, as electrons do not penetrate very far into tissue.

The tissue is infiltrated with the unpolymerised resin, and then heated gently, to polymerise it.

Sections are cut using a glass plate wedge, or a diamond knife on a device called an ultramicrotome.

The cut sections are stained with heavy metal salts. This is because these metals are electron dense, and scatter the electrons. Fewer electrons from these regions reach the film, and on the negative, these regions appear whiter than less electron dense areas. Therefore, this staining increases the contrast of the image. When a print is made from the negative, these regions then appear black. So, on final prints, black areas represent electron dense areas.

Stains commonly used are osmium (as osmic acid), uranium (as uranyl acetate) and lead (as lead hydroxide).

How many sections would you need to cut to sample the entire cell?

questionIf sections are about 100nm thick, and a typical has a diameter of about 20,000nm (20 µm), then how many sections would you need to cut to sample the entire cell?