A technique for labelling the sample surface for quick‐freeze, deep‐etch, rotary replication electron microscopy: application to the study of geletin gel structure

Abstract

A method using magnesium oxide crystals to label the surface of physical gels, such as gelatin gel before quick‐freezing is described and discussed. The quick‐freeze, deep‐etch, rotary replication technique is most adapted to 3‐D visualization of physical gel structure. However, it is known that the depth which ultrarapid freezing may reach is limited by the growth of ice crystals as the distance from the surface of the specimen (rapidly cooled by smashing against a cooled metal plate) increases. Consequently, intact preservation of structures occurs only in superficial zones of the specimen.The MgO surface labelling technique provides a simple means for surface recognition. It enables the estimation of a given replicated area depth, taking into account the angle of specimen scraping before etching and replicating. By comparison of views of the same replica at different depths, freezing artifacts may be recognized even when they cause only slight deformations in the structure. This is particularly necessary for interpretation of gel network geometry: interpretation can be made with certainty only if a reliable surface reference marker exists.For gelatin gels, the depth of best freezing can be estimated to be around 5 μm from the frozen sample surface.