Using high-vacuum evaporation to obtain high-resolution low-temperature images of freeze-fractured membranes from yeast

Abstract

In previous low temperature studies of fractured yeast membranes that were examined in a conventional SEM, Wergin and Erbe demonstrated that resolution could be enhanced by substituting conventional sputter coating of gold with high vacuum evaporation of platinum/carbon. Although this procedure did not clearly resolve membrane particles, the frozen sample could be removed from the SEM and the Pt/C “coat” or replica could be recovered for further examination. Using instruments with greater resolution such as a TEM, as well as a field emission (FE) SEM, to examine the replica did reveal the membrane particles on the replica; therefore the authors concluded that failure to resolve these membrane structures in a conventional SEM probably resulted from limitations of the instrument rather than the coating procedure. Recently, we had the opportunity to interface a FESEM with a cryosystem; this combination was used to determine if samples shadowed in a high vacuum evaporator could be transferred to a cold stage in a FESEM to resolve the macromolecular structure of yeast plasma membranes.