162 5.2  Electron Microscopy

physics and the essential details of the methods in common use and of their applications in

biophysical research laboratories.

5.2  ELECTRON MICROSCOPY

Electron microscopy (EM) is one of the most established of the modern biophysical technolo­

gies. It can generate precise information of biological structures extending from the level of

small but whole organisms down through to tissues and then all the way through to remark­

able details at the molecular length scale. Biological samples are fixed (i.e., dead), and so one

cannot explore functional dynamic processes directly, although it is possible in some cases

to generate snapshots of different states of a dynamic process, which gives us indirect insight

into time-​resolved behavior. In essence, EM is useful as a biophysical tool because the spa­

tial resolution of the technique, which is limited by the wavelength of electrons, in much the

same way as that of light microscopy is limited by the wavelength of light. The electron wave­

length is of the same order of magnitude as the length scale of individual biomolecules and

complexes, which makes it one of the key tools of structural biology.

5.2.1  ELECTRON MATTER WAVES

Thermionic emission from a hot electrode source, typically from a tungsten filament that

forms part of an electron gun, generates an accelerated electron beam in an electron micro­

scope. Absorption and scattering of an electron beam in air is worse at high pressures, and so

conventional electron microscopes normally use high-​vacuum pressures <10⁻³ Pa and in the

highest voltage devices as low as ~10⁻⁹ Pa. Speeds v up to ~70% that of light c in a vacuum (3

× 10⁸ m s⁻¹) can be achieved and are focused by either electromagnetic or electrostatic lenses

onto a thin sample, analogous to photons in light microscopy (Figure 5.1a). However, the

effective wavelength λ is smaller by nearly five orders of magnitude. The difference between

an electron’s rest (E(0)) and accelerated (E(v)) energy is provided by the electrostatic potential

FIGURE 5.1  Electron microscopy. (a) Schematic of a transmission electron microscope.

(b) Typical electron micrograph of a negatively stained section of the muscle tissue (left panel)

showing a single myofibril unit in addition to several filamentous structural features of myofibrils

and a positively shadowed sample of purified molecules of the molecular motor myosin, also

extracted from muscle tissue. (Both from Leake (2001).) (c) Scanning electron microscope (SEM)

module schematic.