Processing a Cryo-Tilt Series with Fiducials

(IMOD 4.9)

University of Colorado, Boulder

 

This is a guided introduction to generating a tomogram from a tilt series of a cryo-sample with gold beads as fiducial markers for alignment.  It presents the most important concepts and details and provides brief explanations of some points.  For more details, consult the Tomography Guide, which you can open from the Help menu in Etomo.  It is also advisable to read through relevant sections of the Tomography Guide before trying to process your own tilt series.  If you have processed a tilt series in Etomo before, you may want to do the dual-axis tutorial before this example, especially if you have difficulty following the steps below without shots of how the screen should appear.

First, a few points on conventions: labels in the Etomo or 3dmod interface are shown in Bold, and entries in fields are shown in italics.  For mouse operations in the Zap window in 3dmod, the buttons are referred to as "first", "second", and "third" because the buttons can be remapped in 3dmod.  If you have not changed the mapping, this corresponds to left, middle, and right; otherwise, it refers to whatever you have chosen to be the first, second, and third buttons.   

Getting started:

 

Tomogram setup:

In this initial step, we define some features of the data set and create the files needed for processing.

Pre-processing:

This step is needed to remove artifacts in the images, generally produced by X-ray events in the camera.  These artifacts will produce streaks in a reconstruction and can also make it harder to see the image features, which have a much smaller dynamic range than the artifacts.

Coarse Alignment

In this step, we use image cross-correlation to align successive images, which makes it easier to track fiducial markers.

Fiducial Model Generation:

In this step, the positions of selected gold markers are found on all of the images, which allows a more accurate alignment to be obtained.

Fine Alignment:

Next the bead positions are fit to a mathematical model of specimen movements.  The model predicts a position for each bead on each view, and the mean distance between the predicted and actual positions is referred to as the "mean residual error".  These errors will let you find and correct badly modeled points.  The need to do so has been much reduced by the recent addition of a method called "robust fitting", which automatically gives less weight or even eliminates the points most likely to be at incorrect positions.  However, it is good to learn how positions can be fixed manually.

Tomogram Positioning

The goal of this step is to set angles and an offset in Z so that the specimen is flat and centered in Z in the computed volume, thus minimizing the computational effort.

 

Final Aligned Stack Creation and CTF Correction:

Gold Erasing:

Gold beads are by far the densest items in cryo-reconstructions and they cast artifactual rays that are about as dense as the biological features.  To minimize this effect, it is often desirable to remove the beads from the projection images before reconstruction.

Tomogram generation:

At last, you can compute the tomogram.

Post-processing:

In this step, you can trim away unneeded regions, convert the tomogram to bytes to save time and space, and reorient the tomogram so that the slices stored in the file are in X/Y planes instead of X/Z planes.  Even if you do not want to trim or convert to bytes, you should always go through this step to get a reoriented tomogram, which will work better with other programs.

Automatic Seed Model Generation:

Making the seed model manually was just an training exercise; you should be able to make this model automatically with almost all of your own data sets.  For an example of doing this, return to the Fiducial Model Generation page.

Clean Up:

In most cases, there is no need for the intermediate files from processing.  This step allows you to remove these files and leave all of the information from which they could easily be recreated if necessary.  The original raw tilt series stack can also "archived" by compressing its difference from the current stack; this operation is reversible.