Tomographic dual tilt series alignment and reconstruction

The aim in aligning a dual tilt series is similar to that of two single tilt series. The difference is that the numbering of the fiducial markers in two series should correspond. The program bdual provides a way to transfer the seed markers from the first to the second series after fitting the zero-tilt micrographs to each other. After tracking the markers and aligning the micrographs, the 3D marker models are fit to each other and the transformations for the second series of micrographs adjusted to correspond to the first, again with the program bdual. A single reconstruction is then calculated from both series.

1. Transferring the seed markers

After both tilt series are set up separately with the correct parameters and normalized micrographs, a fiducial marker seed is selected for the zero-tilt micrograph of the first series. The marker seed is then transfered to the second series:

The argument of the -seed option sets the angular increment (in degrees) to search for the rotation angle that will superimpose the first zero-tilt micrograph onto the second zero-tilt micrograph. The -refine option refines the positions of the markers after the transfer. Each of the tilt series is then tracked and refined separately.

2. Aligning the marker models

After a refined alignment is generated for each tilt series, the 3D marker models are fitted and the micrograph matrices and origins for the second tilt series adjusted to correspond to the framework of the first tilt series:

The -bild output generates a BILD file with the two marker models that can be displayed in UCSF Chimera to assess the quality of the fit. The -Postscript option plots the the x, y and z coordinates of the fitted models to further assess the quality of the fit.

3. Dual tilt reconstruction

Ideally the reconstruction can simply be done as for a single-tilt series, but providing the parameter files for both tilt series:

OR

Alternative, the reconstructions can be done separately, cross-correlated to find the best shift, and combined:

Bernard Heymann 20120126