Improved freeze-etching of difficult specimens

Freeze-etching is now a well established method of biological electron microscopy but, despite its outstanding success in many areas, it still suffers from severe technical limitations when used to study heterogeneous or otherwise intractable tissues. These limitations are mainly related to the difficulty of retaining an intact replica during dissolution of the organic debris, and can be especially acute when dealing with structures composed of many different cell types, or where there are sharp boundaries between hard and soft parts of the specimen. Such difficulties have been described in more detail elsewhere, but they usually have the result that the replica breaks up into small pieces during thawing or cleaning so that it is impossible to locate specific areas of interest.One potential method of improving the retention of intact replicas is to provide an additional supporting layer that can be removed once the organic material has been cleaned away.</jats:p