Bright field electron microscopy of biological specimens—VI Signal-to-noise ratio in specimens prepared on amorphous carbon and graphite crystal supports

Abstract

Three specimen supports (1.3nm and 15nm thick evaporated carbon films and 3–4nm thick graphite crystals) have been used in order to evaluate the gain in signal-to-noise (S/N) ratio in electron micrographs of negatively stained Tobacco Mosaic Virus (TMV) protein. An attempt was also made to obtain images of atoms of selectively stained organic molecules by brigth field electron microscopy. Microdensitometry and optical Fourier analysis were used. For the enhancement of contrast and resolution below 5nm we concluded that the 15nm thick carbon film is not good enough when supporting biological specimens similar to the one used in this investigation. The S/N ratio increased when the TMV protein was supported on 1.3nm thick carbon films. A further improvement was observed when the specimens were prepared on ‘low noise’ graphite supports. If atomic phase contrast is wanted in selectively stained objects, crystal supports must be used. Electron micrographs of a mellitic acid-uranyl acetate complex mixed in the right stoichiometric ratio revaled equilateral projections of triangularly oriented black spots 1.0nm apart. This atomic configuration was difficult to identify amid the defocus dependent granulation of evaporated carbon supports.