Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage.

The term "etching," in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating volage of E = 1 keV and an ion-beam dose of It = 360-400 microA.min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1-2 keV and It = 500 microA.min.