In-lens cryo-high-resolution scanning electron microscopy of large vesicles

High resolution (1-10 nm) imaging of unfixed synthetic vesicles in their na tive, state can be attained using cryogenic temperature high-resolution sca nning electron microscopy. The relatively facile technique involves vitrifi cation of an aqueous suspension of vesicles (similar to 10 mu L) by plunge- freezing in liquid ethane, followed by fracture, coating with a thin layer (1 nm) of Cr, and direct observation of the frozen-hydrated sample on the u pper stage (in-lens) of a field emission scanning electron microscope. A va riety of lipid aggregate morphologies were observed including vesicles with smooth or rough outer surfaces. Membranes were found to fracture either th rough one or both halves of the bilayer, with most vesicles showing a combi nation of the two morphologies.