Ice crystal damage and radiation effects in relation to microscopy and analysis at low temperatures

There are several limitations to the low-temperature techniques which are currently being used for the preparation, examination and analysis of biological and organic samples by means of high-energy beam instrumentation. The low thermal conductivity of samples and the inadequacy of rapid cooling techniques means that, with the exception of thin-film suspensions and the surface of impact-cooled bulk specimens which may be vitrified, ice crystals of varying sizes will be present in nearly all samples which are quench cooled. Data are presented which indicate the depth to which adequate cryo-fixation may be achieved for both morphological and analytical studies. Although dynamic processes may be time resolved in the outer parts of quench-cooled samples, the decreased freezing rate below the surface makes resolution of these processes much less certain. The quality of information which may be obtained from quench-cooled samples is limited by radiation damage. Low-dose microscopy of vitrified thin-film suspensions of macromolecules continues to provide valid structural information at the molecular level. The increased doses needed for X-ray microanalysis present serious problems with the high spatial resolution analysis of thin frozen-hydrated sections although much less damage is observed in dried samples. A case is presented for using the outer fracture faces of frozen-hydrated bulk samples for low-resolution analysis of cells and tissues.