In-Lens Cryo-High-Resolution Scanning Electron Microscopy of Large Vesicles

High resolution (1−10 nm) imaging of unfixed synthetic vesicles in their native state can be attained using cryogenic temperature high-resolution scanning electron microscopy. The relatively facile technique involves vitrification of an aqueous suspension of vesicles (∼10 μL) 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 upper stage (in-lens) of a field emission scanning electron microscope. A variety of lipid aggregate morphologies were observed including vesicles with smooth or rough outer surfaces. Membranes were found to fracture either through one or both halves of the bilayer, with most vesicles showing a combination of the two morphologies.