Methods are being established to characterize the structure and assembly of clathrin triskelions and clathrin cages in vitro to help our understanding of the energetics of receptor-mediated endocytosis. Preparations of the clathrin macromolecular assemblies in various buffers were adsorbed onto thin carbon substrates supported on EM grids, then rapidly frozen in liquid ethane at -180 degrees C. Specimens are either cryotransferred into a high-resolution field-emission STEM for measurement of molecular-weight distributions, or cryotransferred into a vacuum chamber for freeze-etching and platinum/carbon shadowing. It has been necessary to determine the conditions for freeze-drying and metal shadowing to achieve optimal resolution. The metal replicas are imaged digitally in a transmission electron microscope equipped with a slow-scan cooled CCD camera controlled by a Power Macintosh computer. Digital electron microscopy has the advantage of providing immediately accessible data without the need for a dark room.
