Advanced metal coating methods have greatly improved the localization of secondary electrons (SEI) from biological specimens staged in an in-lens field emission SEM. Using these metal coatings, molecular level structural information from cell membranes and isolated bio-molecules have been more accurately recorded from frozen-hydrated samples due to the presence of bound water rather than from chemically fixed and dried samples. Although in-lens cryo-SEM systems have provided images with equivalent resolution to TEM-replicas, several conditions are encountered which require optimizing techniques and hardware.

A drop of hydrated Bakers yeast on a gold Balzers carrier was plunge-frozen in ethane at its melting point. Thermal properties of ethane, such as the large difference between melting and boiling points, and its low viscosity increase the heat transfer coefficient during plunging thereby leading to faster cooling of the sample than with other cryogens. Carriers were transferred to the cryo-workstation of the Oxford CT 3500 system.