DEFECTS OF PLANARITY OF CARBON-FILMS SUPPORTED ON ELECTRON-MICROSCOPEGRIDS REVEALED BY REFLECTED LIGHT-MICROSCOPY

Defects of planarity of two dimensional crystals constitute a major li mitation in high-resolution structural studies of biological macromole cules by electron crystallography. We have found that reflected light microscopy constitutes a simple and appropriate technique for revealin g the state of planarity of carbon films commonly used for supporting specimens in electron microscopy. Carbon films evaporated onto mica an d deposited onto copper grids presented a highly wrinkled aspect. In c ontrast, carbon films prepared after evaporation on nitrocellulose pre sented large areas with an homogeneous flat aspect, while other areas were wrinkled. The wrinkled aspect was also detected by transmission e lectron microscopy at extremely high values of defocusing, by scanning electron microscopy using highly tilted specimens, and was emphasized by Nomarski differential interference contrast in reflected light mic roscopy. We further characterized the surface of carbon films by atomi c force microscopy. The wrinkled aspect was shown to be directly relat ed to the presence of folds extending across carbon films. Typical val ues for the lateral and vertical dimensions of the folds were several micrometers and several hundred nanometers, respectively. These defect s of carbon film planarity correspond to a local tilting of carbon fil ms of the order of 1 degrees. We believe that the wrinkled nature of c arbon films is a general feature of crucial importance for high-resolu tion studies. (C) 1991 Academic Press, Inc.