ANALYSIS OF CARBON-FILM PLANARITY BY REFLECTION-LIGHT MICROSCOPY

We have recently shown that carbon films used as specimen support film s in electron microscopy were highly wrinkled (Schmutz et al., J. Stru ct. 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 pa rallel sets and extending over large distances, Wrinkles were frequent ly observed on one side of these striations. After transfer to electro n microscope grids, carbon films exhibited breaks with a pattern simil ar to that of the striations. In addition, a second type of wrinkle, n ot related to the striations, was observed covering a major surface of every grid, In our attempts to limit the extent of wrinkling, we foun d that the side of electron microscope grids on which carbon films wer e transferred had a preponderant influence on the wrinkling process. T he dull side gave consistently a better result than the shiny side. Th is effect was correlated with scanning electron microscopy observation s. Nitrocellulose films floated on a water surface as well as carbon-c oated nitrocellulose films deposited on electron microscope grids were frequently obtained devoid of breaks and wrinkles, Dissolution of nit rocellulose was accompanied by the apparition of small wrinkles. We al so investigated the wrinkling of carbon-coated grids induced by freezi ng in liquid nitrogen, in relationship with the use of cryo-techniques in electron crystallography of biological macromolecules. Freezing fl at, plain, carbon films resulted in a reversible cryo-induced wrinklin g, in agreement with original observations from Booy and Pawley (Ultra microscopy 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 l ess fragile than plain carbon films.