FREEZING OF AQUEOUS SPECIMENS - AN X-RAY-DIFFRACTION STUDY

The effects on water of two cooling methods, immersion in a liquid cry ogen 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 thic kness. In order to compare the two methods, we mainly studied specimen s having a thickness of 0.2 mm. Under these conditions, freezing by im mersion gives rise to hexagonal (IH), cubic (IC) and amorphous (TV) ic es when the sucrose concentration (weight/weight) has a value within t he range 0-30%, 30-60%, 60% and higher, respectively. The temperature of the phase transitions IV-IC, IC-M depends on the sucrose concentrat ion, High-pressure freezing gives rise to two specific;forms of ice: a n amorphous and a crystalline ice (ice III), Ice III is observed when pure water samples are high-pressure frozen provided that the sample t emperature does not rise above -150 degrees C. Above this temperature, ice III transforms into hexagonal ice, Amorphous ice is formed when t he sucrose concentration is higher than 20%, The amorphous ice formed under high pressure has a similar, but not identical, X-ray diffractio n pattern to that of amorphous ice formed at atmospheric pressure, Whi le the X-ray diffraction pattern of amorphous ice formed at atmospheri c 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 temp eratures that depend on the sucrose concentration, We also observed th at some precautions have to be taken in order to minimize the alcohol contamination of high-pressure frozen samples. The ice-phase diagram p resented in this paper should be taken into account in all methods ded icated to the structural study of frozen biological specimens.