Freeze-etching Technique with Simple Rapid Freezing of Fresh Biological Specimen

In 1957, Steere froze virus crystals, fractured and transferred them to a vacuum evaporator where they were etched, shadowed and coated. The coating, which was then removed from the tissue and mounted on a grid, represented a replica that could be observed in the transmission electron microscope (TEM). This accomplishment is regarded as the first successful biological application of the freeze-etching technique in electron microscopy. This technique was further advanced by describing procedures whereby the replicas from both surfaces of a frozen, fractured sample could be recovered. This pair of replicas provided additional surface information by allowing one to view and compare the two opposing surfaces, or the complementary images, of a freeze-fractured biological specimen. Using a goniometer stage the replicas were also photographed, tilted and rephotographed to produce stereo pairs that could be used to analyze three-dimensionally the surface characteristics of the replica. Until recently these TEM techniques have been the principle procedures utilized by biologists to gain high resolution structural details about virus crystals, biological membranes and a wide range of other material.

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Structures Formed by Cryoprotective Agents Used in Freezc-Etching

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066292 ◽

1971 ◽

Vol 29 ◽

pp. 448-449

Author(s):

G. G. Cocks ◽

C. E. Cluthe

Keyword(s):

Aqueous Solution ◽

Polyethylene Oxide ◽

Liquid Nitrogen Temperature ◽

Ice Crystals ◽

Etching Technique ◽

Structural Constituent ◽

Cryoprotective Agents ◽

Quick Freezing ◽

Freeze Etching ◽

Fractured Surface

The freeze etching technique is potentially useful for examining dilute solutions or suspensions of macromolecular materials. Quick freezing of aqueous solutions in Freon or propane at or near liquid nitrogen temperature produces relatively large ice crystals and these crystals may damage the structures to be examined. Cryoprotective agents may reduce damage to the specimem, hut their use often results in the formation of a different set of specimem artifacts.In a study of the structure of polyethylene oxide gels glycerol and sucrose were used as cryoprotective agents. The experiments reported here show some of the structures which can appear when these cryoprotective agents are used.Figure 1 shows a fractured surface of a frozen 25% aqueous solution of sucrose. The branches of dendritic ice crystals surrounded hy ice-sucrose eutectic can be seen. When this fractured surface is etched the ice in the dendrites sublimes giving the type of structure shown in Figure 2. The ice-sucrose eutectic etches much more slowly. It is the smooth continuous structural constituent surrounding the branches of the dendrites.

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Rapid Freezing of Freeze-Etch Specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003275 ◽

1980 ◽

Vol 38 ◽

pp. 752-755

Author(s):

A. Elgsaeter ◽

T. Espevik ◽

G. Kopstad

Keyword(s):

Low Temperature ◽

Metal Surface ◽

Relative Velocity ◽

Thermal Contact ◽

High Rate ◽

Rapid Freezing ◽

Temperature Decrease ◽

Freeze Etching

The importance of a high rate of temperature decrease (“rapid freezing”) when freezing specimens for freeze-etching has long been recognized1. The two basic methods for achieving rapid freezing are: 1) dropping the specimen onto a metal surface at low temperature, 2) bringing the specimen instantaneously into thermal contact with a liquid at low temperature and subsequently maintaining a high relative velocity between the liquid and the specimen. Over the last couple of years the first method has received strong renewed interest, particularily as the result of a series of important studies by Heuser and coworkers 2,3. In this paper we will compare these two freezing methods theoretically and experimentally.

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Recent progress in the freeze-etching technique

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1971.0042 ◽

1971 ◽

Vol 261 (837) ◽

pp. 121-131 ◽

Cited By ~ 159

Keyword(s):

High Vacuum ◽

Molecular Size ◽

Etching Technique ◽

Structural Distortions ◽

Shadow Casting ◽

Structural Details ◽

Freeze Etching ◽

The One ◽

Insight Into ◽

Made In

The freeze-etching technique must be improved if structures at the molecular size level are to be seen. The limitations of the technique are discussed here together with the progress made in alleviating them. The vitrification of living specimens is limited by the fact that very high freezing rates are needed. The critical freezing rate can be lowered on the one hand by the introduction of antifreeze agents, on the other hand by the application of high hydrostatic pressure. The fracture process may cause structural distortions in the fracture face of the frozen specimen. The ‘double-replica’ method allows one to evaluate such artefacts and provides an insight into the way that membranes split. During etching there exists the danger of contaminating the fracture faces with condensable gases. Because of specimen temperatures below —110 °C, special care has to be taken in eliminating water vapour from the high vacuum. An improvement in coating freeze-etched specimens has resulted from the application of electron guns for evaporation of the highest melting-point metals. If heat transfer from gun to specimen is reduced to a minimum, Pt, Ir, Ta, W and C can be used for shadow casting. Best results are obtained with Pt-C and Ta-W . With the help of decoration effects Pt-C shadow castings give the most information about the fine structural details of the specimen.

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Fine Structure of Swarmers of Cladophora and Chaetomorpha

Journal of Cell Science ◽

10.1242/jcs.9.3.581 ◽

1971 ◽

Vol 9 (3) ◽

pp. 581-601

Author(s):

D. G. ROBINSON ◽

R. D. PRESTON

Keyword(s):

Cell Wall ◽

Fine Structure ◽

Spatial Arrangement ◽

Cell Wall Synthesis ◽

Fibrous Layer ◽

Etching Technique ◽

Freeze Etching

Naked swarmers of both Cladophora rupestris and Chaetomorpha melagonium have been examined by the freeze-etching technique. The swarmers of Cladophora, collected just after settling, reveal several layers of granules external to the plasmalemma and internal to the so-called ‘fibrous-layer’. Chaetomorpha swarmers collected just before settling show extrusion of vesicles through the plasmalemma. The structures associated with the membranes are discussed in relation to known features of these swarmers already observed by sectioning. The role of granules in the synthesis of cell wall microfibrils is strengthened though the spatial arrangement of the granules seen in this investigation does not completely fulfil the ‘ordered granule’ hypothesis. Description of, and comments on, features related to cell wall synthesis, particularly the Golgi and vacuolar systems, are given.

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