Freezing of aqueous specimens: an X-ray diffraction study

The effects on water of two cooling methods, immersion in a liquid cryogen and high-pressure freezing, were studied by X-ray cryodiffraction on different sucrose solutions. The nature of the ice formed by each method depends on both the sucrose concentration and the specimen thickness. In order to compare the two methods, we mainly studied specimens having a thickness of 0.2 mm. Under these conditions, freezing by immersion gives rise to hexagonal (IH), cubic (IC) and amorphous (IV) ices when the sucrose concentration (weight/weight) has a value within the range 0–30%, 30–60%, 60% and higher, respectively. The temperature of the phase transitions IV–IC, IC–IH depends on the sucrose concentration. High-pressure freezing gives rise to two specific forms of ice: an amorphous and a crystalline ice (ice III). Ice III is observed when pure water samples are high-pressure frozen provided that the sample temperature does not rise above ?150 °C. Above this temperature, ice III transforms into hexagonal ice. Amorphous ice is formed when the sucrose concentration is higher than 20%. The amorphous ice formed under high pressure has a similar, but not identical, X-ray diffraction pattern to that of amorphous ice formed at atmospheric pressure. While the X-ray diffraction pattern of amorphous ice formed at atmospheric pressure (IV) shows a broad ring at a position corresponding to 0.37 nm, that of high-pressure amorphous ice (IVHP) shows a broader ring, located at 0.35 nm. IVHP presents a phase transition (IVHP–IV) at temperatures that depend on the sucrose concentration. We also observed that some precautions have to be taken in order to minimize the alcohol contamination of high-pressure frozen samples. The ice-phase diagram presented in this paper should be taken into account in all methods dedicated to the structural study of frozen biological specimens.