Section 19.6.3. Electron scattering and radiation damage

The coherent, elastically scattered electrons contain all the high-resolution information describing the structure of the specimen. The amplitudes and phases of the scattered electron beams are directly related to the amplitudes and phases of the Fourier components of the atomic distribution in the specimen. When the scattered beams are recombined with the unscattered beam in the image, they create an interference pattern (the image) which, for thin specimens, is related approximately linearly to the density variations in the specimen. The information about the structure of the specimen can then be retrieved by digitization and computer-based image processing, as described below (Sections 19.6.4.5 and 19.6.4.6). The elastic scattering cross sections for electrons are not as simply related to the atomic composition as happens with X-rays. With X-ray diffraction, the scattering factors are simply proportional to the number of electrons in each atom, normally equal to the atomic number. Since elastically scattered electrons are in effect diffracted by the electrical potential inside atoms, the scattering factor for electrons depends not only on the nuclear charge but also on the size of the surrounding electron cloud which screens the nuclear charge. As a result, electron scattering factors in the resolution range of interest in macromolecular structure determination (up to  Å⁻¹) are very sensitive to the effective radius of the outer valency electrons and therefore depend sensitively on the chemistry of bonding. Although this is a fascinating field in itself with interesting work already carried out by the gas-phase electron-diffraction community (e.g. Hargittai & Hargittai, 1988), it is still an area where much work remains to be done. At present, it is probably adequate to think of the density obtained in macromolecular structure analysis by electron microscopy as roughly equivalent to the electron density obtained by X-ray diffraction but with the contribution from hydrogen atoms being somewhat greater relative to carbon, nitrogen and oxygen.

Those electrons which are inelastically scattered lose energy to the specimen by a number of mechanisms. The energy-loss spectrum for a typical biological specimen is dominated by the large cross section for plasmon scattering in the energy range 20–30 eV with a continuum in the distribution which decreases up to higher energies. At discrete high energies, specific inner electrons in the K shell of carbon, nitrogen or oxygen can be ejected with corresponding peaks in the energy-loss spectrum appearing at 200–400 eV. Any of these inelastic interactions produces an uncertainty in the position of the scattered electron (by Heisenberg's uncertainty principle) and as a result, the resolution of any information present in the energy-loss electron signal extends only to low resolutions of around 15 Å (Isaacson et al., 1974). Consequently, the inelastically scattered electrons are generally considered to contribute little except noise to the images.

The most important consequence of inelastic scattering is the deposition of energy into the specimen. This is initially transferred to secondary electrons which have an average energy (20 eV) that is five or ten times greater than the valency bond energies. These secondary electrons interact with other components of the specimen and produce numerous reactive chemical species, including free radicals. In ice-embedded samples, these would be predominantly highly reactive hydroxyl free radicals that arise from the frozen water molecules. In turn, these react with the embedded macromolecules and create a great variety of radiation products such as modified side chains, cleaved polypeptide backbones and a host of molecular fragments. From radiation-chemistry studies, it is known that thiol or disulfide groups react more quickly than aliphatic groups and that aromatic groups, including nucleic acid bases, are the most resistant. Nevertheless, the end effect of the inelastic scattering is the degradation of the specimen to produce a cascade of heterogeneous products, some of which resemble the starting structure more closely than others. Some of the secondary electrons also escape from the surface of the specimen, causing it to charge up during the exposure. As a rough rule, for 100 kV electrons the dose that can be used to produce an image in which the starting structure at high resolution is still recognizable is about 1 e Å⁻² for organic or biological materials at room temperature, 5 e Å⁻² for a specimen near liquid-nitrogen temperature (−170 °C) and 10 e Å⁻² for a specimen near liquid-helium temperature (4–8 K). However, individual experimenters will often exceed these doses if they wish to enhance the low-resolution information in the images, which is less sensitive to radiation damage. The effects of radiation damage due to electron irradiation are essentially identical to those from X-ray or neutron irradiation for biological macromolecules except for the amount of energy deposition per useful coherent elastically scattered event (Henderson, 1995). For electrons scattered by biological structures at all electron energies of interest, the number of inelastic events exceeds the number of elastic events by a factor of three to four, so that 60 to 80 eV of energy is deposited for each elastically scattered electron. This limits the amount of information in an image of a single biological macromolecule. Consequently, the 3D atomic structure cannot be determined from a single molecule but requires the averaging of the information from at least 10 000 molecules in theory, and even more in practice (Henderson, 1995). Crystals used for X-ray or neutron diffraction contain many orders of magnitude more molecules.

It is possible to collect both the elastically and the inelastically scattered electrons simultaneously with an energy analyser and, if a fine electron beam is scanned over the specimen, then a scanning transmission electron micrograph displaying different properties of the specimen can be obtained. Alternatively, conventional transmission electron microscopes to which an energy filter has been added can be used to select out a certain energy band of the electrons from the image. Both these types of microscope can contribute in other ways to the knowledge of structure, but in this article, we concentrate on high-voltage phase-contrast electron microscopy of unstained macromolecules most often embedded in ice, because this is the method of widest impact and whose results encompass all resolutions both complementary to and competitive with those from X-ray diffraction.

The important properties of the image in terms of defocus, astigmatism and the presence and effect of amplitude or phase contrast are discussed below (Sections 19.6.4.4 and 19.6.4.6). The best-quality incident electron beam is produced by a field emission gun (FEG). This is because the electrons from a FEG are emitted from a very small volume at the tip, which is the apparent source size. Once these electrons have been collected by the condenser lens and used to produce the illuminating beam, that beam of electrons is then very parallel (divergence of ~10⁻² mrad) and therefore spatially coherent. Similarly, because the emitting tip of a FEG is not heated as much as a conventional thermionic tungsten source, the thermal energy spread of the electrons is relatively small (0.5 to 1.0 eV) and, as a result, the illuminating beam is monochromatic and therefore temporally coherent. Electron beams can also be produced by a normal heated tungsten source, which gives a less parallel beam with a larger energy spread, but is nevertheless adequate for electron cryomicroscopy if the highest resolution images are not required.