Electron microscopy of biological specimens by means of an electrostatic phase plate

Abstract
A new electron microscope imaging method has been developed that is especially suited to the study of thin biological materials. It involves the use of an electrostatic phase plate - a device which creates a more or less uniform difference in optical path between the unscattered and scattered waves by means of its electric field. This phase plate functions in an analogous manner to the absorbing bright contrast phase plate of light microscopy. The contrast effects and aberrations peculiar to the method have been examined and are discussed in terms of their likely influence on the image's representation of the object structure. Analysis of electron micrographs of some biological test specimens, whose structure is relatively well known, confirms that this representation, to a resolution of ca. 0.85 nm, is a particularly faithful one. In the analysis the resolution limit was determined by the degree of specimen preservation, and a real limit, determined by the degree of spherical aberration in the objective lens, of ca. 0.5 nm is expected. A special property of the imaging method, as distinct from the conventional bright field method, is that it emphasizes the detail within the biological material itself, but reduces the contrast from the surrounding film of stain; negative staining remains necessary only because it helps to preserve the morphology of the specimen during irradiation. Evidence is presented that this property enables the method to display information about the specimen that it would not be possible to detect with the bright field method.