Platinum/iridium/carbon: a high-resolution shadowing material for TEM, STM and SEM of biological macromolecular structures.

Thin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze-dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high-resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron-beam-heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre-evaporated. In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength-dispersive X-ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI-layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three-dimensionally stable on the freeze-dried macromolecular samples. By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface-sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze-dried biological structures because of its good electrical conductivity and its direct three-dimensional stability. We compare STM, SEM and TEM images of freeze-dried and Pt/Ir/C-coated polyheads.