Cryo-electron microscopy combined with single-particle reconstruction techniques has allowed us to form a three-dimensional image of the Escherichia coli ribosome.In the interior, we observe strong density variations which may be attributed to the difference in scattering density between ribosomal RNA (rRNA) and protein. This identification can only be tentative, and lacks quantitation at this stage, because of the nature of image formation by bright field phase contrast. Apart from limiting the resolution, the contrast transfer function acts as a high-pass filter which produces edge enhancement effects that can explain at least part of the observed variations. As a step toward a more quantitative analysis, it is necessary to correct the transfer function in the low-spatial-frequency range. Unfortunately, it is in that range where Fourier components unrelated to elastic bright-field imaging are found, and a Wiener-filter type restoration would lead to incorrect results. Depending upon the thickness of the ice layer, a varying contribution to the Fourier components in the low-spatial-frequency range originates from an “inelastic dark field” image. The only prospect to obtain quantitatively interpretable images (i.e., which would allow discrimination between rRNA and protein by application of a density threshold set to the average RNA scattering density may therefore lie in the use of energy-filtering microscopes.
