MATRIX EFFECTS AND THE INDUCTION OF MASS-LOSS OR BUBBLING BY THE ELECTRON-BEAM IN VITRIFIED HYDRATED SPECIMENS

Beam-induced bubbling (i.e. void formation) was studied by cryoelectron microscopy in vitrified thin films of various composition. A differential influence of the cryoprotectant concentration on the beam sensitivity of suspended material (collagen, phospholipid vesicles) was observed; at low cryoprotectant concentrations bubbling started in collagen fibers whereas at high cryoprotectant concentrations (e.g. 40% w/v glycerol) bubbling started at much lower electron doses and collagen fibers and lipid vesicles were affected at approximately the same doses. In vitrified thin films prepared from various concentrations of cryoprotectants bubbling was found to be a function of three parameters: the temperature of cryo-observation, the cryoprotectant concentration and the cooling velocity employed in vitrification. Thus at a given cryoprotectant concentration mass loss dominated at higher temperatures, beam-induced bubbling dominated at low observation temperatures. At a given temperature (e.g. 100 K) mass loss was observed in thin films prepared from low-concentration cryoprotectant (< 20% w/v glycerol), whereas bubbling dominated at higher cryoprotectant concentrations. The effect of the cooling rate was investigated by vitrification of 20% (w/v) glycerol films in liquid nitrogen (''slow'' cooling) or melting ethane (''rapid'' cooling as standard method used in our experiments). In slowly vitrified films bubbling was observed at 100 K whereas only mass loss could be observed at this temperature in the rapidly vitrified films. Since bubbling has also been observed in vitrified films of inorganic material (NaOH, KOH, KCl in aqueous solution) we conclude that the presence of organic material is not essential for the induction of bubbling in cryoelectron microscopy. The packing of water molecules, as influenced by the cryoprotectant concentration, the temperature of observation and the cooling rate during vitrification, is considered as an important parameter in determining the type of beam damage (mass loss or bubbling).