Dual-axis tomography: an approach with alignment methods that preserve resolution.
Tomographic reconstructions of biological specimens are now routinely being generated in our high voltage electron microscope by tilting the specimen around two orthogonal axes. Separate tomograms are computed from each tilt series. The two tomograms are aligned to each other with general 3-D linear transformations that can correct for distortions between the two tomograms, thus preserving the inherent resolution of the reconstruction throughout its volume. The 3-D Fourier transforms of the two tomograms are then selectively combined to achieve a single tomogram. Unlike a single-axis tomogram, a dual-axis tomogram shows good resolution for extended features at any orientation in the plane of the specimen; it also has improved resolution in the depth of the specimen. Calculations indicate that the improvements available from double tilting and from tilting to higher angles are largely additive. Actual and model data were used to assess whether varying the increment between tilted views in proportion to the cosine of the tilt angle would allow a reduction in the number of pictures required to achieve a given resolution of reconstruction. Analysis by Fourier sector correlation indicated that the variable tilt increment improved the reconstruction in some respects but degraded it in others. A varying tilt increment thus does not give an unqualified improvement, at least when using back-projection algorithms for the reconstruction.
Mastronarde
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Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309-0347, USA.
D000818:Animals; D002460:Cell Line; D006056:Golgi Apparatus / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D018386:Kinetochores / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D008962:Models, Theoretical; D051381:Rats; D015203:Reproducibility of Results; D014054:Tomography / Q000379:methods*