Contrast variation in X-ray and neutron scattering

‘Suppose a region [= volume] v of constant shape to be occupied by the molecule which for the present purposes is defined as the region into which salt does not penetrate when a salt solution is substituted for water. It is assumed that the density of the liquid outside v is uniform. The effect on the value of F [= structure factor] of increasing density everywhere outside v is equivalent to that of decreasing density by an equal amount everywhere inside v, since a uniform distribution of density has no effect on F. It follows that the values F(water) F(salt) are the F values for a region v of uniform density equal to the difference between the electron concentration of the salt solution and water’. This argument was introduced by Bragg & Perutz (1952) to explain reversible changes in the intensities of low-order X-ray reflections from a hemoglobin crystal and to deduce from these the shape and orientation of the hemoglobin molecule occupying the volume v within the unit cell. It also the description of what became later known as contrast variation in small-angle scattering from macromolecules in solution (Stuhrmann & Kirste, 1965). Contrast variation by solvent exchange is a low-resolution method. It is for this reason it was been abandoned in protein crystallography a few years after its introduction in 1952. In fact, the introduction of small-sized labels – one (or very few) heavy metal atoms per protein molecule – led to be the first determination of a protein structure at nearly atomic resolution (Kendrew et al., 1960). About ten years later, a similar development started in small-angle scattering with the introduction of specifically deuterated macromolecules in neutron small-angle scattering (Engelman & Moore, 1972). Somewhat earlier, in the late sixties, discussions went on about the visualization of single polymer chains in the bulk by deuteration using neutron scattering. For this purpose dilute solid solutions of the ordinary polymer in the perdeuterated polymer had to be prepared (Kirste, 1970). The study on vitreous polymethylmethacrylate showed that the unperturbed random coil is the conformation of chain molecules in the amorphous solid state (Kirste et al., 1972). A number of similar studies have been published since then. A first collection of these was presented at the Third International Conference on X-ray and neutron small-angle scattering in 1973. While the above mentioned methods are still used, new methods of contrast variation in both X-ray and neutron scattering have come up in the last twenty years. This paper will focus on anomalous or resonant contrast and nuclear spin contrast. 2. Anomalous or resonant contrast