Sample size: an important parameter in flash-cooling macromolecular crystallization solutions

The effect of sample size on flash-cooling was assessed using an assortment of Hampton Screen I solutions ( Hampton Research). These were cryogenically cooled to ascertain the minimum glycerol concentration needed for vitrification of solutions. Solutions were 'flash-cooled' in 1.0, 0.5 and 0.1 mm commercial cryoloops, using a 100 K nitrogen stream generated by an Oxford 600 Cryostream. The loops were visually examined and then X-ray diffraction data were collected and evaluated for ice formation. The glycerol concentration needed for vitrification was found to decrease significantly as sample size decreased; detailed key data are presented in tabular form. Glass transition temperatures, T-g, were measured for solutions leading to successful vitrification in the 1 mm cryoloop. The trends in the required concentration of glycerol and resultant T-g can be understood through a simplified heat-transfer analysis. This analysis points to a critical cooling time period for successful vitrification.