Preparation of flat carbon support films

Wrinkling of carbon support films is known to limit the resolution of electron microscopy images of protein two-dimensional crystals. The origin of carbon wrinkling during preparation of the support films was investigated by reflected light microscopy. We observed that carbon films go through several states during their preparation. While dried carbon films have a tendency to be wrinkled, a flat state is observed transiently before complete drying. This state could be stabilized by the addition of sugars or tannic acid to the embedding medium. An alternative method for preparing flat carbon films was developed, in which a sandwich is formed by two symmetrical carbon films positioned on both sides of a grid. The formation of sandwiched carbon films was facilitated by the use of grids with thin bars. The carbon sandwich films were flat, stable, and easily and reproducibly prepared.