Concentration of solutes during preparation of aqueous suspensions for cryo-electron microscopy

It is demonstrated that solutes are likely to be significantly concentrated under conditions commonly used to prepare vitrified aqueous suspensions for transmission electron microscopy. Muscle thick filaments in such suspensions were largely dissolved, probably due to an increase in salt concentration caused by evaporation of water immediately prior to freezing. The extent of solubilization indicated that salts had been concentrated by at least 50%. Simple tests showed that, under the conditions used, the rate of reduction in the height of a thin water layer was approximately 1 micron/s. Apparatus is described which reduces evaporation, by clamping the hydrated grid in a filter paper sandwich until just before it enters the ethane coolant. High-speed cine photography showed that, using this device, exposure of the thinned specimen to the atmosphere was approximately 200-fold less. Frozen-hydrated thick filaments prepared using the device had the expected length of about 1.6 microns.