Section 19.2.2. The electron microscope

An electron microscope is conceptually analogous to a light microscope. It consists of an electron source, condenser lenses, an objective lens, projector lenses and a camera recorder. Because of the electronegative property of the electron, it is possible to fabricate magnetic and electrostatic lenses to focus electrons to near atomic resolution. The most critical lens in an electron microscope is the objective lens, which forms the first diffraction pattern at its focal plane and the first image at its image plane. An electron diffraction pattern is the same as an X-ray diffraction pattern, containing only the amplitude information of the structure factor, whereas an electron image contains both the amplitude and phase information of the structure factor (Unwin & Henderson, 1975).

The condenser lens is used primarily to control the beam diameter and the flux of the electrons irradiating the specimen, whereas the projector lenses are used to magnify either a diffraction pattern or an image in a broad range. The camera length in an electron microscope is adjustable, ranging from 0.2 to 2.5 m, and allows the recording of diffraction patterns with Bragg spacings from hundreds to a fraction of an ångstrom. The magnification of an image spans from a hundred to a million times. Magnification is not a limiting factor for image resolution, however, and is typically set between 40 and 80 000 times for protein electron crystallography. The most important factors that affect the instrumental resolution are the coherence of the incident electron beam, the chromatic and spherical aberrations of the objective lens, the electrical stability of the electron gun and the objective lens, and the mechanical stability of the specimen stage (Chiu & Glaeser, 1977). In general, almost all modern electron microscopes are capable of resolving a lattice spacing of 2.4 Å in a thin gold crystal. The biological structure resolution in an image of a protein crystal has not reached the instrumental resolution because of many factors related to radiation damage of the specimen. Improvements in experimental and computational methods, however, have made it feasible to image protein crystals beyond 3.7 Å resolution (see below).

The range of electron energy used in an electron microscope is between 20 and 1000 keV, which corresponds to wavelengths of 0.086–0.0087 Å. Any single microscope is built and optimized for a narrow energy range because of the complex design of a highly stable electron gun. The instrument most commonly used for molecular-biology structure research operates in the range 100 to 400 keV. Choosing the most useful electron energy is based on the desired resolution and the specimen thickness. The thicker the specimen, the higher the energy that should be used in order to avoid dynamical scattering effects and to have a sufficient depth of field, so that the electron scattering data can be interpreted with a single scattering theory. Theoretically, the specimen thickness should not exceed about 700 Å if the targeted resolution is 3.5 Å with a 400 kV microscope. Beyond this specimen thickness, the phase error of the structure factors might approach 90° at that resolution. An added advantage of using higher electron energy is to reduce the chromatic aberration effect, resulting in a better-resolved image (Brink & Chiu, 1991).

There are different types of electron emitters, including tungsten filaments, LaB₆ crystals and field emission guns, all of which use different mechanisms to generate the electrons. The field emission gun produces the brightest, most monochromatic beam. The high brightness can allow the electrons to be emitted as from a point source to irradiate the specimen with a highly parallel (i.e. a highly spatially coherent) illumination. The benefit of high spatial coherence is the preservation of high-resolution details in the image, even though the defocus of the objective lens is set very high in order to have low-resolution feature contrast (Zhou & Chiu, 1993). Thus, a field emission source is the best choice for high-resolution data collection.

The recording medium of an electron microscope can be an image plate, a slow-scan charge-coupled-device (CCD) camera, or photographic film. Because of their broad dynamic range and high sensitivity, both the CCD camera and the image plate are best suited for recording diffraction patterns (Brink & Chiu, 1994). However, for high-resolution image recording – when the recorded area, pixel resolution, signal-to-noise ratio and the modulation transfer function characteristics must be considered – photographic film is the optimal choice (Sherman et al., 1996).