Considerations for tilt series alignment in IMOD

When doing the tilt series alignment in IMOD there are a few things we strongly recommend to follow in order to reach the best possible results.

Alignment parameters

To avoid overfitting, the tilt parameters should describe a rigid body transformation, i.e., we only solve for the parameter “one rotation”. The parameter selection should look as follows:

Alignment parameters in IMOD for rigid body transformation. We only solve for one rotation and keep everything else fixed

Residual fixing

In the “residual fixing” step in IMOD, the residuals should be fixed individually but only in a way that makes sense, i.e., the centers of the model (green circles) should always be in the center of the gold beads, even if the residual (red arrow) points somewhere else. This can be achieved by manually moving/correcting the green circles (right mouse click) if necessary.

Removing outlier beads

If there are outlier beads in your model that keep having large residuals no matter how well centered the model is, they can be removed from the model. However, this should only be done in extreme cases. Make sure you keep enough gold beads in your final model (minimum 5, ideally 10-15 or as many as behave well).

Alignment error

The alignment error should preferably be below 2 pixels and in the best cases below 1 pixel. However, this should not be forced. As said before, keep the points of the model on the center of the beads and keep enough gold beads in the model. It can be that some tomograms simply cannot be aligned better.

Alignment quality

Once you are satisfied with the alignment you can quickly check the alignment quality by confirming that the beads are moving perfectly horizontal throughout the tilts. Drawing a “red box” with the corresponding tool in 3dmod can be helpful as a visual guide.

Tomogram quality

Another way to check the alignment quality is by inspecting the beads in the final tomogram. A good alignment results in perfectly symmetric beads (fig. 3).