The resolution achieved in low-dose electron microscopy of biological
macromolecules is significantly worse than what can be obtained on the
same microscopes with more robust specimens. When two-dimensional
crystals are used, it is also apparent that the high-resolution image
contrast is much less than what it could be if the images were perfect.
Because specimen charging is one factor that might limit the contrast
and resolution achieved with biological specimens, we have investigated
the use of holey support films that have been coated with a metallic
film before depositing specimens onto a thin carbon film that is
suspended over the holes. Monolayer crystals of paraffin
(C44H90) are used as a test specimen for this
work because of the relative ease in imaging Bragg spacings at ∼0.4
nm resolution, the relative ease of measuring the contrast in these
images, and the similar degree of radiation sensitivity of these
crystals when compared to biological macromolecules. A metallic coating
on the surrounding support film does, indeed, produce a significant
improvement in the high-resolution contrast for a small fraction of the
images. The majority of images show little obvious improvement,
however, and even the coated area of the support film continues to show
a significant amount of beam-induced movement under low-dose
conditions. The fact that the contrast in the best images can be as
much as 25%–35% of what it would be in a perfect image is
nevertheless encouraging, demonstrating that it should be possible, in
principle, to achieve the same performance for every image. Routine
data collection of this quality would make it possible to determine the
structure of large, macromolecular complexes without the need to grow
crystals of these difficult specimen materials.