X-rays are generated when charged particles interact with an electromagnetic field. For the purposes of x-ray crystallography, x-ray generation is provided by the acceleration of electrons. The first x-ray tubes involved a heated cathode and an anode sealed in a vacuum. Electrons generated at the cathode are accelerated toward the anode target; the ensuing deceleration in the target gives rise to a continuum of x-rays ('white radiation', also known as Bremsstrahlung) (Figure 12). The minimum wavelength as well as the intensity distribution of the rays is dependent upon the accelerating voltage. In addition, a series of intense spectral lines is superimposed upon the 'white' background that is characteristic of the nature of the anode target used. These lines correspond to the excitation of inner electrons in the target by bombarding electrons of sufficient energy; relaxation of the excited electrons back to their ground state results in quantized x-ray production of defined wavelength. The most commonly used target for protein crystallography is copper, whose strongest spectral line (Cu Ka radiation, resulting from the L to K energy level transition) has a wavelength of 1.5405A. Molybdenum, whose short Mo Ka wavelength of 0.7093A interacts only weakly with biological macromolecules, is more often used by small-molecule crystallographers, while chromium has attracted interest in recent years as the Cr Ka wavelength of 2.29 aA is suitable for a wider range of anomalous scattering techniques (see Section 18.104.22.168.2).38,39
Clearly, bombardment of an anode with high-energy electrons leads to serious problems of heat dissipation. While so-called sealed tube generators with stationary targets are relatively easy to maintain and to align, the heating problem presents obvious limitations to the maximum intensity obtainable. As described above, the interaction of x-rays with biological matter is particularly weak. In order to overcome this, rotating anodes have been developed, which now represent the workhorse of most in-house sources used for structural biology. For particularly small or weakly scattering crystals, however, the use of synchrotron radiation has become indispensable. In a synchrotron, relativistic electrons (or positrons) are held in a closed orbit by way of a series of bending magnets. The centripetal acceleration experienced by the electrons toward the center of the ring results in an intense beam of white electromagnetic radiation, ranging from infrared to hard x-rays (Figure 13). Higher intensities can be obtained using so-called insertion devices such as wigglers and undulators: multipole magnets inserted into straight sections of the electron beam path between bending magnets that allow a controlled deflection of the electrons.
For nearly all single crystal projects, monochromatic radiation is required. In former times, this was achieved by placing a filter in the beam path: for Cu radiation, for example, a nickel filter will absorb most of the Cu Kb radiation, leaving a relatively clean Cu Ka beam. According to Bragg's law, a well-defined and precisely oriented crystal will allow only a single wavelength to pass through. Applying a slight curvature to a crystal mirror allows focusing and collimation of the x-ray beam to a certain extent, so that most modern x-ray sources are fitted with perpendicular mirror optics to focus the beam in the horizontal and vertical directions. More recently, mirrors coated with graded multilayers have become available that can provide higher flux or lower beam divergence. Continuing advances in x-ray optics have opened the way to microfocus x-ray sources with reasonable x-ray flux yet lower running costs. Monochromators are particularly important for MAD and SAD experiments carried out at synchrotrons - to be able to collect a reliable anomalous data set at the absorption edge, it is necessary to have a precision in wavelength of the order of 0.0001 A (corresponding to 2 eV; Figure 13).
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