X-ray crystallography has for a long time provided scientists with an insight into structure at an atomic level. As the structures become more complicated, DNA for example, image construction becomes more difficult. Taking advantage of so-called anomalous diffraction has successfully simplified this problem, but this required a periodic sample, so biological molecules had to be assembled into a crystal structure. Now, scientists from the USA, Germany and France have shown that it is possible to use anomalous diffraction to image non-periodic structures (Phys. Rev. Lett. 101, 076101; 2008).

Credit: © 2008 APS

The difficulty in applying X-ray diffraction techniques to molecules made up of thousands of atoms — macromolecules — stems from the increasing number of diffracted and reflected rays. Not only the amplitudes of all of these rays need to be measured to construct a useful image, but also the phase, a technical challenge often referred to as the 'phase problem'. Multiple-wavelength anomalous diffraction (MAD) reduces the problem from one with many different types of atom to one that involves just one or two that scatter X-rays in an unusual way. The information gained in this way serves as a reference that provides an insight into the structure of the entire molecule.

Anomalous diffraction occurs when the energy of the incident photons is resonant with an electron transition in a metallic atom, leading to the scattering of light. Anomalous scattering differs from normal scattering in that, as it is based on a resonance, it is very wavelength-dependent (but almost independent of scattering angle). By probing the sample with different wavelength X-rays, wavelength-dependent and wavelength-independent elements of the diffraction pattern can be separated. This approach has proved a boon in structural biology, and can even be applied to macromolecules that have no metallic content, by introducing the appropriate heavy metal atoms.

Andreas Scherz and co-workers have now shown that this technique can even be used on samples that don't exhibit any periodicity. To prove this, they fabricate a test sample made up of 300-nm- and 90-nm-diameter polystyrene spheres on an aperture in a gold film 1.2 μm across. This target was then exposed to spatially coherent X-rays (about 106 photons s−1μm−2) from the Stanford Synchrotron Radiation Laboratory for between 700 s and 1,000 s. Two wavelengths were chosen near the edge of the 'K' absorption line of carbon, one quite close to the resonance and the other a little further away (wavelengths of about 4.4 nm). The interference of the diffraction patterns at these two wavelengths holds the key to creating an image. An iterative computer algorithm was used to disentangle the phase information, and in this way the team is able to image the spheres with a resolution of approximately 20 nm.

The authors say their technique could be applied to both organic and inorganic nanostructures, enabling investigations of, for example, chemical composition or magnetic behaviour.