Analysis of carbon film planarity by reflection light microscopy

We have recently shown that carbon films used as specimen support films in electron microscopy were highly wrinkled (Schmutz et al., J. Struct. Biol. 112 (1994) 252). We report here on a further analysis of the planarity of carbon films at the different steps of their preparation, by reflection light microscopy. Carbon films evaporated on mica and floated on a water surface presented straight striations aligned in parallel sets and extending over large distances. Wrinkles were frequently observed on one side of these striations. After transfer to electron microscope grids, carbon films exhibited breaks with a pattern similar to that of the striations. In addition, a second type of wrinkle, not related to the striations, was observed covering a major surface of every grid. In our attempts to limit the extent of wrinkling, we found that the side of electron microscope grids on which carbon films were transferred had a preponderant influence on the wrinkling process. The dull side gave consistently a better result than the shiny side. This effect was correlated with scanning electron microscopy observations. Nitrocellulose films floated on a water surface as well as carbon-coated nitrocellulose films deposited on electron microscope grids were frequently obtained devoid of breaks and wrinkles. Dissolution of nitrocellulose was accompanied by the apparition of small wrinkles. We also investigated the wrinkling of carbon-coated grids induced by freezing in liquid nitrogen, in relationship with the use of cryo-techniques in electron crystallography of biological macromolecules. Freezing flat, plain, carbon films resulted in a reversible cryo-induced wrinkling, in agreement with original observations from Booy and Pawley (Ultramicroscopy 48 (1993) 273). No cryo-induced wrinkling was noticed with carbon-coated nitrocellulose films, while wrinkling was observed after nitrocellulose dissolution, although these films were significantly less fragile than plain carbon films.