Figure 9–40 The resolution of the electron microscope. this transmission electron micrograph of a monolayer of graphene resolves the individual carbon atoms as bright spots in a hexagonal lattice. graphene is a single isolated atomic plane of graphite and forms the basis of carbon nanotubes. the distance between adjacent bonded carbon atoms is 0.14 nm (1.4 Å). such resolution can only be obtained in a specially built transmission electron microscope in which all lens aberrations are carefully corrected, and with optimal specimens; it cannot be achieved with most conventional biological specimens. (From a. Dato et al., Chem. Commun. 40:6095–6097, 2009. With permission from the royal society of Chemistry.)
The source of illumination is a filament or cathode that emits electrons at the top of a cylindrical column about 2 m high. Since electrons are scattered by collisions with air molecules, air must first be pumped out of the column to create a vacuum. The electrons are then accelerated from the filament by a nearby anode and allowed to pass through a tiny hole to form an electron beam that travels down the column. Magnetic coils placed at intervals along the column focus the electron beam, just as glass lenses focus the light in a light microscope. The specimen is put into the vacuum, through an airlock, into the path of the electron beam. As in light microscopy, the specimen is usually stained—in this case, with electron-dense material. Some of the electrons passing through the specimen are scattered by structures stained with the electrondense material; the remainder are focused to form an image, in a manner analogous to the way an image is formed in a light microscope. The image can be observed on a phosphorescent screen or recorded with a highresolution digital camera. Because the scattered electrons are lost from the beam, the dense regions of the specimen show up in the image as areas of reduced electron flux, which look dark.
In the early days of its application to biological materials, the electron microscope revealed many previously unimagined structures in cells. But before these discoveries could be made, electron microscopists had to develop new procedures for embedding, cutting, and staining tissues.
Since the specimen is exposed to a very high vacuum in the electron microscope, living tissue is usually killed and preserved by fixation—first with glutaraldehyde, which covalently crosslinks protein molecules to their neighbors, and then with osmium tetroxide, which binds to and stabilizes lipid bilayers as well as proteins (Figure 9–42). Because electrons have very limited penetrating power, the fixed tissues normally have to be cut into extremely thin sections (25–100 nm thick, about 1/200 the thickness of a single cell) before they are viewed. This is achieved by dehydrating the specimen, permeating it with a monomeric resin that polymerizes to form a solid block of plastic, then cutting the block with a fine glass or diamond knife on a special microtome. The resulting thin sections, free of water and other volatile solvents, are supported on a small metal grid for viewing in the microscope (Figure 9–43).
Figure 9–41 The principal features of a light microscope and a transmission electron microscope. these drawings emphasize the similarities of overall design. Whereas the lenses in the light microscope are made of glass, those in the electron microscope are magnetic coils. the electron microscope requires that the specimen be placed in a vacuum. the inset shows a transmission electron microscope in use. (photograph courtesy of JeoL Ltd.)
Figure 9–42 Two common chemical fixatives used for electron microscopy.
the two reactive aldehyde groups of glutaraldehyde enable it to cross-link various types of molecules, forming covalent bonds between them. osmium tetroxide forms cross-linked complexes with many organic compounds, and in the process becomes reduced. this reaction is especially useful for fixing cell membranes, since the C=C double bonds present in many fatty acids react with osmium tetroxide.