For “Fiducial Model Gen. Not Started”:

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

2. Look for “Total number” under “Seed Points to Select, and put 50 in the blank (this number depends on how many gold beads are in your tomogram, we have a lot here)

   1. This function can automatically select the number of gold beads (the dark and round spots you see in the tomogram) you want. I put 50 because I can see more than 50 gold beads in my tomogram and they distribute nicely. Judge based on your tomogram.

3. Click “Generate Seed Model” and wait. This function automatically selects gold beads at 0 degree tilt angle.

4. Click “Open Seed Mode”

5. Three new windows will pop up. It’s from an incorporated software called 3dmod.

   First window allows you to visualize the frames.

   

   Second window allows you to see the functions of 3dmod.

   

   Third window allows you to delete and add points, which are gold beads in this case.

   1. Make sure the “Operation” selects “Make seed”

      

   2. Make sure “Autocenter” is checked.

6. Now you need to evaluate the gold beads the software automatically selected

   1. Delete a point

      1. if it is near the edge of the image, probably will disappear at high tilt angles, so you can delete it.
      2. if a bunch of gold beads aggregates together, delete them as the software later can’t distinguish
      3. if the gold beads are under/near some impurity (such as ice contamination, dark grid), delete it as the software may not distinguish the right location of the beads.
      4. if the function selects a point where there is no gold bead.

      

      Aggregated gold beads are not selected.

      

      Near-edged gold beads are not selected.

      

   2. How to delete

      1. Select the point/circle in the first window by click it with the left button on your mouse.
      2. Go to the second window, Click “Edit”, Go to “Contour”, Click “Delete”
      3. Shortcut: Shift+D

   3. Add a point if

      1. if you feel you have too few points, and there are some that haven’t been automatically selected.
      2. if you feel there are some really good points on the edge or in an empty area

   4. How to add

      1. Go to the first window
      2. Hover your mouse pointer to where the gold bead (point) you want to add.
      3. Click with the scroll wheel

   5. Move a point if

      1. The circle doesn’t align with the gold bead at their centers, although there is a gold bead.

   6. How to move

      1. Select the point in the first window by click it with the left button on your mouse.
      2. Hover your mouse pointer to where the gold bead (point) you want to move.
      3. Click that place with the right button on your mouse.

   7. Warning: In the second window, make sure you must have 1 point for each contour. If not, you will see an error when you do the next step in “Track Seed Model”

   8. How to save if you modified anything at this step

      1. Go to the second window, Click “File”, Click “Save Model”
      2. Shortcut: S
      3. If you forget, there should be a pop-up window asking “Save model before quitting?”

   9. Go to the second window, and click “X” on the right up corner to close 3dmod.

7. Now you are back to the etomo window where you told the software how many gold beads you want it to automatically pick.

8. Click “Track Beads” and you will be directed to a new window. This window will automatically track the gold beads the software selected at 0 degree tilt angle and propagate it to all the frames. The goal is to make sure every frame has the same number of points, selected gold beads.

9. Click “Track Seed Model”

10. At this point, you should notice you have another small rectangular-shaped window called “Project Log”. We need to use it extensively during the following steps. If not, go to etomo window, hover your pointer to “View”, and click “Show/Hide Log Window”. Shortcut: Ctrl+L.

11. At the Project Log, check the number of “Total points missing = “

    1. if = 0, you can proceed to step 14
    2. else if > 100, the selected gold beads probably have some big problems and you need to carefully check
    3. else if <100, some gold beads might be missing at high tilt angles.

    

12. Click “Fix Fiducial Model”

13. You now see 3 windows identical to the 3 windows you see at Step 5 of this section.

14. Make sure, in the third window, instead of “Make seed” was selected as step 5, “Fill gaps” is selected this time.

15. Make sure “Autocenter” is checked as well.

16. Click “Go to Next Gap”. This automatically directs you to the contour where gold beads are missing (check the second window) and the frame before/after the frame (check the first window) that is missing a gold bead to be selected.

    1. You can also check the number of points for that contour. If the number is well smaller than that of the other contours, you can just delete this contour with Shift+D.

17. You now should see in the first window, a yellow circle with an arrow appeared.

    1. If the arrow points upward, it means the frame after this frame is missing a gold bead to be selected.
    2. If the arrow points downward, it means the frame before this frame is missing a gold bead to be selected.
    3. You can go to the frame after with “PageUp” on your keyboard, or the frame before with “PageDown”. Or you can manually adjust the frame number in the first window at the place in the image below.

    

18. After you arrive at the frame that is missing one gold bead, find which unselected gold bead in this frame is the same gold bead as the one in the previous frame.

    1. if you can’t find it, you can always delete that “Contour” with Shift+D.

19. Hover your pointer to that gold bead, make sure “Autocenter” is checked, and click it with the scroll wheel on your mouse, to add it to the point list (check the second window, you will see the total number of points of that specific contour is increased by one).