4.3 Electron Microscopy: High-Resolution Imaging
Electron microscopic imaging of biological samples, such as single proteins, organelles, cells, and tissues, offers a much higher resolution of ultrastructure than can be obtained by light microscopy. The short wavelength of electrons means that the limit of resolution for a transmission electron microscope is theoretically 0.005 nm (much less than the diameter of a single atom), or 40,000 times better than that of a light microscope and 2 million times better than that of the unaided human eye. However, the effective resolution of the transmission electron microscope in the study of biological systems is considerably less than this ideal. Under optimal conditions, a resolution of 0.10 nm can be obtained with transmission electron microscopes, about 2000 times better than with conventional light microscopes and at least 300 times better than with the current super-resolution microscopes.
The fundamental principles of electron microscopy are similar to those of light microscopy; the major difference is that in electron microscopes, electromagnetic lenses focus a high-velocity electron beam instead of the visible light used by optical lenses. In the transmission electron microscope (TEM), electrons are emitted from a filament and accelerated in an electric field (Figure 4-28, left). A condenser lens focuses the electron beam onto the sample; objective and projector lenses focus the electrons that pass through the specimen and project them onto a viewing screen or other detector. Because atoms in air absorb electrons, the entire tube between the electron source and the detector is maintained under an ultrahigh vacuum. Thus living material cannot be imaged by electron microscopy.
FIGURE 4-28 In electron microscopy, images are formed from electrons that pass through a specimen or are scattered from a metal-coated specimen. In a transmission electron microscope (TEM, left), electrons are extracted from a heated filament, accelerated by an electric field, and focused on the specimen by a magnetic condenser lens. Electrons that pass through the specimen are focused by a series of magnetic objective and projector lenses to form a magnified image of the specimen on a detector, which may be a fluorescent viewing screen, a photographic film, or a charged-couple-device (CCD) camera. In a scanning electron microscope (SEM), electrons are focused by condenser and objective lenses on a metal-coated specimen. Scanning coils move the beam across the specimen, and electrons scattered from the metal are collected by a photomultiplier tube detector. In both types of microscopes, because electrons are easily scattered by air molecules, the entire column is maintained at a very high vacuum.
In this section, we describe various approaches to viewing biological material by electron microscopy. The most widely used instrument is the transmission electron microscope, but also in common use is the scanning electron microscope (SEM), which provides complementary information, as we discuss at the end of this section (Figure 4-28, right).