Enhanced contrast in electron microscopy of unstained biological material

Abstract

In-focus phase contrast electron microscopy has been investigated for the enhancement of bulk contrast (i.e. the contrast of large regions) of model biological specimens. Carbon film phase plates, of measured thickness, were introduced into the back focal plane ofthe objective lens. Image contrast was determined from Faraday-cage intensity measurements. A contrast enhancement was observed but was measured to be less than that obtained using a very small objective aperture. This was attributed to the smaller proportion of elastic scattering and the limited spatial frequency region over which the phase contrast transfer function was uniform. Electron beam interferometry established the ability of the phase plates to preserve the coherence of the beam traversing them. Carbon granularity, of specific dimensions, was significantly enhanced by the phase plate in accordance with the phase contrast transfer function and this enhanced granularity dominated the images of biological specimens. I N T R O D U C T I O NThe phase contrast technique, invented for light microscopy by Zernike (1942a, b), involved phase shifting the scattered relative to the non-scattered waves from a microscope specimen. Constructive or destructive interference was achieved when the wave components interfered in the image plane and image contrast was enhanced by rendering a specific detail brighter (negative contrast) or darker (positive contrast) than its surroundings. A phase plate in the diffraction plane (back focal plane) of the objective lens accomplished the required phase shift by providing an increased optical path (a phase delay) for one wave component relative to the other.Calculations (Bennet et al., 1946; Franqon, 1950;Barer, 1952) indicated the necessity of a 7r/2 rad phase delay of scattered waves for positive phase contrast in the case of hypothetical objects comparable to typical unstained biological specimens. Experimentally, Zernike (1942a, b) and Bennet et al. (1946, 1951) found a 57/2 rad phase plate to be the best compromise for routine biological light

High voltage electron microscopy of wet whole cells

“…As was anticipated, dark field gave very poor resolution and contrast on thick specimen areas (Johnson & Parsons, 1969) but improved contrast and equal resolution for thin areas. This can be seen in Fig.…”

High resolution dark‐field electron microscopy

“…for very thin specimens) that dark-field image formation produces high contrast. Numerical data obtained from theoretical and practical measurements (Hall, 1951;Johnson & Parsons, 1969) showed that this plateau is situated in the region of 40-60 nm gm/cm3 for an acceleration voltage in the order of 100 kV. This mass thickness includes the supporting film.…”

Aspects of dark‐field electron microscopy

“…( 2 ) Theoretical study (a) Di'erence from strioscopy. The contrast-stop method is often called strioscopy (Johnson & Parsons, 1969). Such a term may suggest wrong analogies.…”