Metal Shadowing and Decoration in Electron Microscopy of Biological Macromolecules

This symposium coincides with the fortieth anniversary of the introduction of shadowing, the use of an evaporated heavy metal film to impart contrast in electron microscopy.'Since its inception, the basic method has changed little, although improvements in instrumentation and a fuller knowledge of the physics involved have led to a gradual increase in reproducibility and quality.24Slayter recently presented in these Annals an account of the various landmarks in the evolution of shadowing from the perspective of the fibrinogen s t r ~c t u r e .~ Two technical advances are particularly significant-the development of the electron gun for evaporating high-melting-point metals and the availability of quartz crystal film thickness monitors.It is perhaps surprising that the use of electron beams to evaporate high-melting-point material was implemented some fifteen years ago,6 and yet has been widely practiced only relatively recently.The majority of the thousands of biological applications of shadowing that have been published during the last twenty-five years have been associated with two quite separate methodological developments-the surface spreading technique for nucleic acids (and other negatively charged polymers)' and techniques of freeze-fracturing and free~e-etching.'.~ Electron microscopy for nucleic acid analysis is now being replaced in many instances by more convenient biochemical techniques, but for many years it has been the most reliable way of sizing DNA, detecting A-T rich regions, mapping heteroduplex molecules, and detecting spliced segments in mRNA."Its requirements for shadow quality are relatively undemanding.In addition to stabilizing the molecules at an air-water interface, the envelope of cytochrome c a t least doubles the effective diameter of DNA, so that the fibers are readily visualized at low shadowing angles even when the metal grains are large by high-resolution standards.Thus, although higher-resolution methods for visualizing nucleic acids in their naked state either through shadowing" or positive staining'* have been developed, there has been little incentive to adopt them due to the adequacy of simpler techniques.The introduction of freeze-fracturing and related methods, for which shadowing is the sole contrasting method, has played a decisive role in cell biology, especially in understanding membrane structure.Prior to the first freeze-fracture results most knowledge of cellular ultrastructure was based on thin sectioning, and there was always the disturbing possibility that the "alchemy" involved in fixation, dehydration, and embedding might introduce gross artifacts.