For “Fine Alignment Not Started”:

1. You will see another very complicated window. Take a breath.

2. Look for “Minimization Parameters” and check “Do robust fitting with tuning factors: 1.0”

3. Look for “Local Alignment Parameters”, check “Enable local alignments”, and choose “# of local patches (x,y): 5,5”.

4. Click “Compute Alignment” and wait

5. Check your “Project Log” and Look for the values of “Residual error local mean” and “Residual error weighted mean”

   1. Ideally, we would only continue if “Residual error local mean” is smaller than 0.3 and “Residual error weighted mean” is smaller than 0.4 (In this dataset, the majority would have a residual error local mean around 0.3, so anything significantly higher, such as 0.5, would signify some problems, such as noise, of your tomograms. However, if in general your dataset has a local mean around 0.5, then this would be your ideal threshold)
   2. But for WT_24, you see it’s 0.332 and 0.497, respectively.
   3. We will continue for the sake of this tutorial

   

6. Go back to the First etomo window

7. Click “View/Edit Fiducial Model”, and 3 windows will pop up.

8. Click “Go to Next Big Residual” on the window that is vertically long.

9. Then on the window that shows you the tomogram, you should see a green circle has another yellow circle pops up. This is the point that the software thinks had big residual, meaning not at the right location. Note, the software is not always right. You need to judge if the point is at the center of a gold bead with your eye. You can always zoom in to view the location better (by click up and down arrows in the first window).

   1. If you feel it’s already at the center, click “Go to Next Big Residual”
   2. If you feel it’s not, move your mouse pointer to where you think it should be, click that place with the right button on your mouse. Tah-Dah, your point is moved.
      1. Alternatively, you can click “Move Point by Residual”, which the software moves the point to where it thinks it should be.
   3. To speed up this process, the Shortcut for “Go to Next Big Residual” is the apostrophe button on the keyboard, and “Move Point by Residual” is the semicolon button.

10. If you delete a contour and got an error in the second window like this: “Point is > 15 pixels from the position in log file. You need to save the model file, close the 3 windows, and re-run “Compute Alignment”

—You can skip the part below—

1. Save the model as before, or press S on the keyboard. Close the 3dmod window.

2. Use your mouse to right click on the main interface at an empty spot

3. You will see a list like this appear (try different place if you can’t get it open)

   

4. Click on “Align log file” and you will see a new window like this pops up

   

5. Go to this new “Align” window and click “Solution”

   1. Check if “mean resid” is smaller than 1

6. Then click “Surface Angles”

7. Check if “Incremental tilt angle change” is smaller than 0.1