Supersaturation patterns in counter-diffusion crystallisation methods followed by Mach–Zehnder interferometry

Abstract

We present experimental observation of the spatio-temporal pattern of supersaturation in counter-diffusion methods. These complex patterns were recorded by dynamical interferometric analysis using a Mach–Zehnder configuration. Tetragonal hen egg white lysozyme crystals were grown inside APCF (advanced protein crystallisation facility) reactors. Salt and protein diffusion profiles were obtained independently by performing duplicated experiments with and without protein in the protein chamber; salt gradients were observed directly while protein concentration profiles are computed from the differences in refractive index between the two experiments. As expected from computer simulations, the time evolution of supersaturation shows a maximum about 45 h after activation (although this value can change as a function of the starting conditions and the geometry of the reactor). Nucleation takes place before this maximum supersaturation is reached. This explains the trend of the growth rate versus time curves for experiments performed within APCF reactors (both on ground and in space) and in capillaries by the gel acupuncture technique. By using very low concentration agarose gel in the protein chamber, sedimentation and buoyancy effects are eliminated so that the effects of gravity on fluid dynamics and hence on the spatio-temporal evolution of supersaturation can be assessed. These results confirm experimentally the predicted behaviour of counter-diffusion systems and support their use in growing large high-quality protein single crystals.