X-ray diffraction to probe the kinetics of ice recrystallization inhibition

Understanding the nucleation and growth of ice is crucial in fields ranging from infrastructure maintenance, to the environment, and to preserving biologics in the cold chain. Ice binding and antifreeze proteins are potent ice recrystallization inhibitors (IRI), and synthetic materials that mimic this function have emerged, which may find use in biotechnology. To evaluate IRI activity, optical microscopy tools are typically used to monitor ice grain size either by end-point measurements or as a function of time. However, these methods provide 2-dimensional information and image analysis is required to extract the data. Here we explore using wide angle X-ray scattering (WAXS/X-ray powder diffraction (XRD)) to interrogate 100's of ice crystals in 3-dimensions as a function of time. Due to the random organization of the ice crystals in the frozen sample, the number of orientations measured by XRD is proportional to the number of ice crystals, which can be measured as a function of time. This method was used to evaluate the activity for a panel of known IRI active compounds, and shows strong agreement with results obtained from cryo-microscopy, as well as being advantageous in that time-dependent ice growth is easily extracted. Diffraction analysis also confirmed, by comparing the obtained diffraction patterns of both ice binding and non-binding additives, that the observed hexagonal ice diffraction patterns obtained cannot be used to determine which crystal faces are being bound. This method may help in the discovery of new IRI active materials as well as enabling kinetic analysis of ice growth.