Cryo - Fesem of Subcellular Organisation Using an In-Lens Rod with A Vacuum Transfer System

Abstract

The recent increase in use of Field Emission Scanning Instruments in biology has shown the potential of these systems for sub-cellular imaging, as they essentially provide transmission EM levels of resolution for surface imaging. in structures lacking inherent contrast in thin sections, such as nuclear pore complexes,(NPC) high resolution surface imaging has provided significant complementary structural information to the macromolecular architecture of the NPC. (Goldberg and Allen, 1996). A series of intermediate structural forms of the NPC during its formation has also been described using FESEM, which would not have been resolvable in TEM.(Goldberg, Weise, Allen, Wilson, 1997). All this work was performed with chemical fixation, dehydration and critical point drying, followed by high resolution Chromium coating. We now are extending these studies to a cryo-preservation approach, mindful of the requirement of rapid freezing to preserve the more labile events of membrane dynamics, and also to circumvent the requirement to expose membranes to organic solvents during conventional dehydration.