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Tilt-Series Alignment

Since this data set has gold fiducials, we could use bead tracking for manual tilt series alignment using software like the Etomo program in IMOD. Manual alignment often yields superior results to automated alignment, but for sake of time we will use TOMOMAN to perform automated tilt-series alignment using AreTomo. TOMOMAN provides some additional settings on top of the standard AreTomo settings, including the option to use unfiltered or dose-filtered tilt-series, as well as pre-binning the tilt-series prior to alignment.

For more information about AreTomo, see the publication or manual.

Open tomoman_aretomo.param.

  1. The directory parameters block should already be set correctly.

  2. The AreTomo files and folders block should already be set correctly. We will generate a bin8 tomogram so bin8_aretomo/ is a good output directory name.

  3. The AreTomo parameters block determines how AreTomo will be run.

    1. Set InBin to 4. We find that processing the dose-filtered stack with an input binning of 4 yields better results.

    2. AlignZ and VolZ are measures of thickness along the z axis measured in unbinned pixels. AlignZ is the thickness of the temporary volume used for alignment. This should be approximately the same as the actual thickness of the sample. VolZ is the thickness of the reconstructed tomogram and should be larger than both AlignZ and the specimen thickness. In this case, AlignZ=1400 and VolZ=1800 should work well. The rest of the AreTomo parameters can be left as defaults.

    3. Check that OutBin is set to 8. This is the final binning, so it is effectively a additional 2x binning from the 4x binning of our input tilt-series.

    4. Use the same Gpu setting as for motion correction.

  4. Run reconstruction as before in the TOMOMAN console.

    tomoman(pwd,'tomoman_aretomo.param');

You now have a reconstructed tomogram you can visualize in 3dmod.

3dmod bin8_aretomo/TS_01_dose-filt_bin8.mrc

In the 3dmod window, select Image > XYZ to (or use the shortcut Ctrl + X) view slices along all axes.

NOTE: After reconstruction, you should see that the tomogram is reconstructed beyond the edges of the specimen along the Z direction. This is evidence that the 'AlignZ' and 'VolZ' parameters we set were appropriate.