The origins and evolution of freeze-etch electron microscopy.

The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future.

Direct observations of freeze-etching processes of ice-embedded biomembranes by atomic force microscopy.

We have fabricated a cryogenic atomic force microscope that is designed for structural investigation of freeze-fractured biological specimens. The apparatus is operated in liquid nitrogen gas at atmospheric pressure. Freeze-fracturing, freeze-etching and subsequent imaging are carried out in the same chamber, so that the surface topography of a fractured plane is easily visualized without ice contamination. A controlled superficial sublimation of volatile molecules allows us to obtain three-dimensional views of ultrastructures of biological membranes.

Design and application of a freeze-fracture stage with a cleaver device for Balzers 'freeze-etching' apparatus.

A fracturing mechanism in the form of a double-wedged cleaver, used previously on other freeze-fracture machines, has been incorporated in the Balzers BAF 400 D apparatus. The design of the stage, with the cleaver, is explained and its performance and advantages are outlined. By minimizing mechanical damage to the exposed surface of the frozen specimen and reducing contamination by effective shrouding, an improvement in the quality of the replicas is achieved. The suitability of this mechanism for the fracturing of tough tissues such as skin is also outlined.

An improved specimen table for the Balzers freeze etching system BAF 400.

A time saving, simple and inexpensive adaptation of a Balzers specimen table is described. This specimen table is used in combination with the small specimen holders originally developed for the Denton Freeze-etching module. They are in particular suited for cell suspensions, and are easy to handle for freezing procedures. Ten replicas are produced routinely per freeze fracture run, with the aid of this combined system.

In situ liquid propane jet-freezing and freeze-etching of monolayer cell cultures.

A procedure for in situ liquid propane jet-freezing of unfixed and unglycerinated monolayer cell cultures is described. We have used the method to study monolayer cultures of rat Sertoli cells and human monocytes. The described procedure gives a cooling rate during specimen freezing high enough to yield adequately frozen specimens even in the absence of any cryoprotectant. Use of the procedure therefore eliminates artefacts usually caused by cell fixation and addition of cryoprotectives.

Some methodical aspects and applications of freeze-etching in polymer research.

The freeze-etching method is used for investigating the morphology of aqueous polymer systems, e.g. network-like segregation structures of cryofixed polymer solutions and real network structures in polymer gels. The cooling rate, which is decisive for cryofixation of specimens, is measured by a thermocouple and a pyroelectric element in different coolants. The effect of the cooling rate on the freeze-etching structure of polymer solutions, polymer gels and a 10% aqueous glycerol solution as a model substance is determined and compared. The increase of the cooling rate in polymer gels results in maintaining the original network structure, in smaller segregation compartments in polymer solutions and in smaller crystallites in the cryofixed ice matrix.

A simple freeze-fracturing and etching apparatus using the cold block principle.

An inexpensive freeze-fracturing and etching equipment, based on the cold block principle, is described. The specimen is fractured inside the apparatus before replication with the aid of a simple fracturing device. The cleavage plane can be preselected and complementary fracture surfaces of tissue blocks are easily obtained. Since fracturing is carried out without the use of knife, large areas can be studied on the replica which is free of knife marks. Temperature measurement and heating of the lower block makes controlled etching possible. The apparatus can be constructed in a laboratory workshop at very reasonable expense; the results have been comparable to those obtained with most good quality or sophisticated freeze-etching equipments.

Freeze-etching of dispersions without contamination of the fracture faces.

The physical processes occuring while the fracture face of a dispersion is being freeze-etched are described and the resulting opportunities for elimination of contamination phenomena are discussed. An anticontamination device which can be used in combination with a drop freeze-fracture apparatus is described. The main advantages of the device, high effectiveness and great simplicity, are demonstrated by several practical examples.

A resistance monitor with power cut-off for automatic regulation of shadow and support film thickness in freeze-etching and related techniques.

A resistance monitor with sensors and automatic power cut-off has been developed to control the thickness of Pt-C shadow and C replica films in freeze-etching and related techniques. The monitor and sensors, in conjunction with newly modified evaporators, should considerably reduce the amount of C and Pt required and should prove useful in other applications employing vacuum evaporation of thin films of Pt, C, or other conducting materials.