The SEM technique provides great depth of field; moreover, since the amount of electron scattering depends on the angle of the surface relative to the beam, the image has highlights and shadows that give it a threedimensional appearance (see Figure 9–49 and Figure 9–51). Only surface features can be examined, however, and in most forms of SEM, the resolution attainable is not very high (about 10 nm, with an effective magnification of up to 20,000 times). As a result, the technique is usually used to study whole cells and tissues rather than subcellular organelles (see Movie 21.3). Veryhighresolution SEMs have, however, been developed with a bright coherentfield emission gun as the electron source. This type of SEM can produce images that rival the resolution possible with a TEM (Figure 9–52).
negative staining and Cryoelectron Microscopy Both allow Macromolecules to Be Viewed at high resolution
If they are shadowed with a heavy metal to provide contrast, isolated macromolecules such as DNA or large proteins can be visualized readily in the electron microscope, but negative staining allows finer detail to be seen. In this technique, the molecules are supported on a thin film of carbon and mixed with a Figure 9–49 A developing wheat flower, solution of a heavymetal salt such as uranyl acetate. After the sample has dried, or spike. this delicate flower spike was rapidly frozen, coated with a thin metal film, a very thin film of metal salt covers the carbon film everywhere except where it and examined in the frozen state with an has been excluded by the presence of an adsorbed macromolecule. Because the seM. this low-magnification micrograph macromolecule allows electrons to pass through it much more readily than does demonstrates the large depth of focus of the surrounding heavymetal stain, a reverse or negative image of the molecule is an seM. (Courtesy of kim Findlay.) created. Negative staining is especially useful for viewing large macromolecular aggregates such as viruses or ribosomes, and for seeing the subunit structure of protein filaments (Figure 9–53).
Shadowing and negative staining can provide highcontrast surface views of small macromolecular assemblies, but the size of the smallest metal particles in the shadow or stain used limits the resolution of both techniques. An alternative that allows us to visualize directly at high resolution even the interior features of threedimensional structures such as viruses and organelles is cryoelectron microscopy, in which rapid freezing to form vitreous ice is again the key. A very thin (about 100 nm) film of an aqueous suspension of virus or purified macromolecular complex is prepared on a microscope grid and is then rapidly frozen by
Figure 9–50 The scanning electron microscope. in an seM, the specimen is scanned by a beam of electrons brought to a focus on the specimen by the electromagnetic coils that act as lenses. the detector measures the quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen and controls the intensity of successive points in an image built up on a screen. the seM creates striking images of three-dimensional objects with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument. (photograph courtesy of andrew Davies.)