Freeze-fracture cytochemistry: a simplified guide and update on developments

Abstract

A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.