Enhanced contrast in electron microscopy of unstained biological material I. Strioscopy (dark-field microscopy)

Electron-optical methods of enhancing contrast are being investigated in order to eliminate the artifacts associated with heavy-metal contrasting procedures. This report gives a quantitative discussion of the application of the strioscopic dark-field method to model biological objects. Beam-stopping apertures were constructed by microwelding fine platinum wires on to platinum objective apertures. These were precisely located and centred in the back focal plane of the objective lens. Contrast was evaluated by a sensitive micro-Faraday cage system. Bright-field and strioscopic contrast of organic layers up to 557 nm thickness were investigated and related to the transition from single to plural scattering. The spherical-aberration phase-contrast contribution could be separated from the amplitude contrast in bright-field microscopy by extrapolation to zero object thickness. Strioscopic contrast of thin layers was found to be much greater than bright field aperture contrast and shown to be non-linear with respect to thickness in the region 0.7-10 nm. For diffraction patterns made up of a few discrete beams the resolution is high, but is considerably less for diffusely scattering and thick objects. Strioscopy may be the method of choice for obtaining contrast of unstained or weakly-stained objects at high acceleration voltage.