Freeze-Etching Electron Microscopy: Recent Developments and Application to the Study of Biological Membranes and Their Components

A survey of ultra‐rapid cryofixation methods with particular emphasis on applications to freeze‐fracturing, freeze‐etching, and freeze‐substitution

“…Other findings on model systems are the following: Whereas conventionally frozen water/egg-PC suspensions (more than 30% egg-PC) depict ice crystal damage in the form of irregular textures of ice-bilayer pockets, such damage is absent in rapidly-frozen suspensions, where large and smooth lipid bilayer leaflets are separated by ice pockets. In combination with X-ray diffraction observations, these findings suggest that, here too, rapid-freezing preserves the initial sample structure adequately (Gulik-Krzywicki, 1985;Gulik-Krzywicki and Costello, 1978). Also, it is better to examine small and large unilamellar vesicles and liposomes with sprayfreezing than with other electron microscope preparation techniques.…”

“…Structural rearrangements are less likely to occur in membranes of rapidly-frozen than in those of conventionally (i.e., chemically fixed and/or cryoprotected before freezing) treated specimens. This is especially the case for lipid systems, where high temperature phases (e.g., Costello et al, 1981;Ververgaert et al, 1973; and samples with a high water content (Gulik-Krzywicki and Costello, 1978;Gulik-Krzywicki, 1985) can be adequately retained for study (Section We), as well as for lipid-protein systems, where less rearrangement of membranebound particles occurs (Section IVf; e.g., Morel et al, 1980;Ornberg and Reese, 1979;Thompson et al, 1985). Also, improvement of the rapid-freeze technology facilitates study of inner and outer membrane surfaces (e.g., Espevik and Elsgaeter, 1981a;Heuser and Salpeter, 1979;Shohet et al, 1981) (Section IVf).…”

“…Rapid-and high pressure-freezing prior to any other preservational treatment provide a superior means of preservation for ultrastructural or micro-probe analysis compared to preservational techniques that involve chemical fixation or chemical fixation combined with cryoprotection before cryofixation. This applies to replica, as well as to whole mount and sectioning (including freeze-substitution) techniques (Dubochet et al, 1981;Dudek and Boyne, 1986;Escaig, 1982a;Gilkey and Staehelin, 1986;Gulik-Krzywicki, 1985;Gupta and Hall, 1984;Hohling and Krefting, 1984;Humber and Muller, 1984;Hunziker and Schenk, 1984a,b;Meister and Muller, 1980;Metz, 1981;Plattner, 1984;Plattner and Bachmann, 1982;Plattner and Zingsheim, 1983;Rash, 1983;Robards and Sleytr, 1985;Shotton, 1980;Sitte, 1982;Sitte and Neumann, 1985;Umrath, 1977;Van Venetie, 1982;Zeitler, 1982). However, at the time that most of those reviews were written there were only a limited number of publications on freeze-fracturing, -etching, and-substitution with rapid-and high pressure-freezing.…”

“…Under very high vacuum conditions (pressure < 10pgmbar) at very low temperatures (-260°C) Pt/C shadowing also gives a fine grain replica (Gross et al, 1985). Additional exploration of Ta/W evaporation might further improve on the direct observation of the substructure of macromolecules with freeze-etch techniques (Gross et al, 1985;Heuser, 1983b;Gulik-Krzywicki, 1985). In order to evaporate Ta/ W at acceptable rates, the thermal characteristics of the tungsten rod on which the Ta/ W alloy is formed, require water cooling at the rod-holder.…”

“…Fujikawa (1980Fujikawa ( , 1981a offers an explanation for this phenomenon. On the basis of his studies on erythrocytes he suggests that in regions with ice crystals underneath, the membrane can be rather disordered as a consequence of compression (see also Gulik-Krzywicki, 1985;Gulik-Krzywicki and Costello, 1978). In such regions, the membrane no longer displays a proper bilayer configuration.…”