Prospects for using an IVEM with a FEG for imaging macromolecules towards atomic resolution.

Specimen preparation and imaging techniques for biological macromolecules have been improved to the point where attention to the electron-optical imaging conditions becomes a significant factor for achieving high resolution. A field emission gun (FEG) can provide an illumination source with a better spatial and temporal coherence suitable for imaging near atomic resolution. Our computational analysis of carbon film images taken between Scherzer focus and 1.1 microns underfocus (20x Scherzer focus) with the Hitachi 200 kV microscope with a cold field emission gun shows detectable contrast beyond 3.5 A resolution. In biological imaging, a large defocus is often used to optimize the low-resolution contrast in order to facilitate the subsequent steps in computer reconstruction. An intermediate-voltage electron microscope (IVEM) would optimize the contrast at high resolution by reducing the temporal coherent effects. In theory, the IVEM would give a greater depth of field so that large macromolecular assemblies such as viruses and cellular structures can be interpreted and reconstructed reliably using the projection approximation. These experimental and theoretical considerations provide a rationale for designing a future IVEM with a FEG suitable for biological macromolecule imaging close to atomic resolution.