Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryoelectron tomography.

Using a 400-kV cryoelectron microscope, we have obtained tomographic reconstructions of frozen-hydrated sea urchin axonemes with 8-10-nm resolution, as assessed by detection of characteristic components including doublet microtubules, radial spokes, central sheath projections, and outer dynein arms. We did not detect the inner dynein arms or the microtubule lattice. The 1/(8 nm) and 1/(16 nm) layer lines are consistently present in power spectra of both projection images and tomographic reconstructions. Strength and detection of the layer lines are dependent upon total electron dose and defocus. Both layer lines are surprisingly resistant to electron doses of up to 11000 electrons/nm(2). We present a summary of resolution considerations in cryoelectron tomography and conclude that the fundamental limitation is the total electron dose required for statistical significance. The electron dose can be fractionated among the numerous angular views in a tomographic data set, but there is an unavoidable fourth-power dependence of total dose on target resolution. Since higher-resolution features are more beam-sensitive, this dose requirement places an ultimate limit on the resolution of individual tomographic reconstructions. Instrumental and computational strategies to circumvent this limitation are discussed.