High-Vacuum Evaporation and a Cryostage to Observe Fractured Yeast

Abstract

Investigators have long realized the potential advantages of using a low temperature (LT) stage to examine fresh, frozen specimens in a scanning electron microscope (SEM). However, long working distances (W.D.), thick sputter coatings and surface contamination have prevented LTSEM from achieving results comparable to those from TEM freeze etch. To improve results, we recently modified techniques that involve a Hitachi S570 SEM, an Emscope SP2000 Sputter Cryo System and a Denton freeze etch unit. Because investigators have frequently utilized the fractured E face of the plasmalemma of yeast, this tissue was selected as a standard for comparison in the present study.In place of a standard specimen holder, a modified rivet was used to achieve a shorter W.D. (1 to -2 mm) and to gain access to the upper detector. However, the additional height afforded by the rivet, precluded use of the standard shroud on the Emscope specimen transfer device. Consequently, the sample became heavily contaminated (Fig. 1). A removable shroud was devised and used to reduce contamination (Fig. 2), but the specimen lacked clean fractured edges. This result suggested that low vacuum sputter coating was also limiting resolution.