TITLE: FEI Spirit Direct Alignments
AUTHORS: Mason Mackey, NCMIR Lab

[DESCRIPTION]
The protocol outlines the step-by-step procedure for direct alignment of the FEI Spirit microscope, ensuring optimal performance for electron microscopy. The process includes specimen holder setup, high tension adjustment, and alignment of various components such as the beam, gun tilt, condenser astigmatism, and objective aperture. Specific attention is paid to maintaining eucentricity and optimizing beam shift and focus for high-resolution imaging.

[GUIDELINES]
Ensure proper specimen handling and alignment to avoid damaging the electron microscope.
Maintain a clean and controlled environment for microscopy operations.
Follow each alignment step carefully to avoid misalignment, which can affect the quality of imaging.

[STEPS]
SECTION: Alignment Steps
20. Beam shift alignment. Go to the cross-over using the “Intensity” knob on the left panel. Cross-over is the smallest beam spot possible. Click “Beam Shift” in the Direct Alignments. Using multifunction (MF) X-Y knobs, adjust to center the beam on the viewing screen. Click “Done” button. Do this after each alignment. 
SECTION: Alignment Steps
21. Gun tilt alignment. Click “Gun Tilt” in the Direct Alignments. Using one MF knob at a time, adjust the intensity of the beam to its maximum (Smallest exposure time). Click “Done” button.
SECTION: Alignment Steps
22. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click “Done” button.

SECTION: Alignment Steps
23. Condenser aperture alignment (not on screen, above objective aperture). Manually select an aperture size with the outermost aperture knob (1 through 4). 1 is the smallest and 4 the largest. Use the “Intensity” knob and spread the beam clockwise to see if the beam spreads evenly across the viewing screen. If the beam expands and contracts symmetrically, then the aperture is centered. If the beam sweeps off the screen, then adjust the two aperture knobs until the beam stays centered.  
SECTION: Alignment Steps
24. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click “Done” button.
SECTION: Alignment Steps
25. Condenser Astigmatism. Select “Stigmators” in the popup panel (right-bottom corner of the UI). Choose “Condenser”. Use the multifunction (MF) knobs to do the correction. For the condenser, spread the beam to half the diameter of the viewing screen. Adjust the beam shape to become a circle.  Then click “None” when finished.

SECTION: Alignment Steps
26. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click “Done” button.

SECTION: Alignment Steps
27. Gun shift alignment. Use the L3 programmable button to set the spot size to 3. Select “Gun Shift”. Use MF knobs to bring the beam to the center of the screen. Switch to the spot size 9 (R3 programmable button). Select Align beam shift. Use MF knobs to bring the beam to the center of the screen. Repeat until the beam is in the center for both spot sizes 3 and 9. Bring back to spot size 1 when finished. 

SECTION: Alignment Steps
28. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click the “Done” button.

SECTION: Alignment Steps
29. Beam tilt pivot point X and Y alignment. Click “Beam tilt pp X” in the Direct Alignments. The beam will begin to wobble. Using MF knobs, stop the beam wobbling, so that beam is stationary in the center of the screen. Click “Done” button. Select “Beam tilt pp Y” in Direct Alignments. Repeat the above procedure for the Y pivot points. Click “Done”.

SECTION: Alignment Steps
30. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click the “Done” button.

SECTION: Alignment Steps
31. Objective aperture alignment. Press “Diffraction”. Manually select an objective aperture size (1 through 4). 1 is the smallest and 4 the largest. Center the aperture using the two aperture knobs until the aperture is centered over the diffraction spot (not the center of the viewing screen). After aligning, press “Diffraction” to turn off diffraction mode.

SECTION: Alignment Steps
32. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel to go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click the “Done” button.

SECTION: Alignment Steps
33. Rotational center alignment. Use high magnification (23Kx or 26Kx) for this alignment. Perform this alignment on a visible feature (region of interest) on the sample that is centered on the viewing screen. Turn the focus knob counterclockwise (underfocus) and see the direction shift of the visible feature. If the feature is moved, select “Rotation Center” alignment in the Direct Alignments. Image of the feature will start wobbling in and out. Use MF knobs to center visible feature. Click the “Done” button and press “Eucentric Focus”. Repeat this alignment again to check if the feature is centered when focusing. 

SECTION: Alignment Steps
34. Go back to magnification 9700X. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel, go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click the “Done” button.

SECTION: Alignment Steps
35. Repeat objective aperture alignment. Press “Diffraction”. Center the aperture using the two aperture knobs until the aperture is centered over the diffraction spot. After aligning, press “Diffraction” to turn off diffraction mode.

SECTION: Alignment Steps
36. Cross-over and beam shift alignment. Use the “Intensity” knob on the left panel, go to cross-over and click “Beam Shift” in the Direct Alignments. Using MF, adjust to center the beam on the viewing screen. Click the “Done” button.

SECTION: Alignment Steps
37. Gain reference.  

SECTION: Alignment Steps
38. Objective Astigmatism.

SECTION: Set up
1. Put dewar in holder and make sure copper cold fingers are inserted.  
SECTION: Set up
3. Remove specimen holder:
SECTION: Set up
3.1. Before removing the holder, make sure the column valves are closed.
SECTION: Set up
3.2. In the Search tab located in Workset, align the stage in the middle by double clicking the center of the grid.
SECTION: Set up
3.3. Place your left fingers on the goniometer purple flange and grab the holder with your right hand.
SECTION: Set up
3.4. Pull the holder straight back until it stops and make a small clockwise rotation. (holder at this point will not go back in the column)
SECTION: Set up
3.5. Adjust your hand and keep turning clockwise until it stops.
SECTION: Set up
3.6. Use your right thumb to push on the purple flange and take the holder straight out.
SECTION: Set up
4. Put the holder in the clear protective stand and remove the cap to reveal the specimen holder tip.
SECTION: Set up
5. While holding the handle with your hand, place the tool pin into the hole in front of the spring clamp,  lift the specimen clamp upwards to 90 degrees, remove the tool pin, replace the tool pin in the stand and insert the grid (dark side facing down). With the same tool pin, place it in the hole and close the clamp downwards while holding the handle. Place the tool pin back into the stand.
SECTION: Set up
6. Remove the holder from the protective stand.
SECTION: Set up
7. Insert specimen holder:
SECTION: Set up
7.1. Carefully orient the large guide pin at the 11 o’clock position & the small airlock pin at the 5 o’clock position, insert the holder as far as it can go. Slightly push to make sure it is in all the way.
SECTION: Set up
7.2. Select ‘single tilt’ and press the arrow next to the rectangle box just above the SA magnification.
SECTION: Set up
7.3. Wait until the pumping is finished. (Pumping count down timer reaches zero in the vacuum overview).
SECTION: Set up
7.4. Once the stage red LED light is off, immediately turn the holder counter clockwise. Make sure to hold the holder tightly while inserting because the vacuum strongly pulls the holder in. Do not let go until it is secured inside.
SECTION: Set up
8. High Tension set up
SECTION: Set up
8.1. Start at 20Kv
SECTION: Set up
8.2. Press ‘High Tension’ and wait for the emission to go down to 10.
SECTION: Set up
8.3. Set to 40Kv
SECTION: Set up
8.4. Wait for the emission to go down to 10.
SECTION: Set up
8.5. Set to 60Kv
SECTION: Set up
8.6. Wait for the emission to go down to 10.
SECTION: Set up
8.7. Set to 80Kv
SECTION: Set up
8.8. Wait for the emission to go down to 10.
SECTION: Set up
9. Turn on Filament (takes approximately five minutes)
SECTION: Set up
9.1. Log on book. Wait until the filament heats to 30 degrees.??? double check
SECTION: Magnification Set up
10. Set magnification to SA 9700X
SECTION: Magnification Set up
11. Set Spot Size to 1 by pressing L3 and R3 on control pad.
SECTION: Magnification Set up
12. Set Focus Step to 5 
SECTION: Magnification Set up
13. Open Col. Valves. 
SECTION: Before Alignment
14. Before doing direct alignments one must set eucentricity to set lenses to factory set. Press “Eucentric Focus”.
SECTION: Before Alignment
15. Choose a window that is not broken by surfing through the simple with the stage trackball.
SECTION: Before Alignment
16. Select a latex bead and center it with the stage trackball.
SECTION: Before Alignment
17. Press L2 “Alpha Wobble'' and press Z + or – to set eucentricity (the bead barely moves and is within the inner circle). Press L2 again to turn off “Alpha Wobble”.
SECTION: Before Alignment
18. Press L1 to set defocus to zero. (This tares the  defocus to zero where the focal point is at the specimen. Focal point is the point at which parallel rays of electron converge at and diverge at from the specimen)
SECTION: Alignment Steps
19. *All alignments except astigmatism are accessible via the Direct Alignments Control panel in the “Workset” or from the popup panel.*
SECTION: Alignment Steps
37.1. Open SerialEM.
SECTION: Alignment Steps
37.2. Take a pencil and place it between the specimen holder end and the goniometer. (One does not want a specimen in the beam path).
SECTION: Alignment Steps
37.3. Go to (Camera & Macro Controls) and open “Setup”.
SECTION: Alignment Steps
37.4. Make sure “Record” exposure time is set to 1 second at bin 1, “Focus” exposure time is set to 0.2 second at bin 2 and “View” exposure time is set to 0.2 second at bin 2.
SECTION: Alignment Steps
37.5. Press “OK”.  Place the focus screen in the viewing chamber and spread the beam clockwise to exposure time 0.40s on the “Set Up” page in FEI software.
SECTION: Alignment Steps
37.6. Go to the “Camera” pull down menu on Serial EM and select “ Prepare Gain Reference”. 
SECTION: Alignment Steps
37.7.  Make sure “Target number of counts” is set at 1800 and “Number of Frames” is set to either 5 or 10.
SECTION: Alignment Steps
37.8. Press “Yes” to spot position aligned. (This means the electron beam is aligned).
After gain reference, press “Record” to check image gain reference image. 
SECTION: Alignment Steps
37.9. Remove the pencil. 
SECTION: Alignment Steps
38.1. Place the focus screen in the viewing chamber and spread the beam to exposure time 0.40s on the “Set Up” page in FEI software. 
SECTION: Alignment Steps
38.2. In Serial EM, press “View” in Camera & Macro Control. 
SECTION: Alignment Steps
38.3. Go to the “Process” pull down menu in Serial EM and select “Live FFT”.
SECTION: Alignment Steps
38.4. Slightly defocus using the focus knob counterclockwise to spread the FFT ring. 
SECTION: Alignment Steps
38.5. Go to “Stigmators” at the popup panel (right-bottom corner of the UI).
SECTION: Alignment Steps
38.6. Choose “Objective”. Use the multifunction (MF) knobs to do the correction (make sure the ring is circular/isotropic).
SECTION: Alignment Steps
38.7. Press “None” when finished correcting for objective astigmatism.
SECTION: Alignment Steps
38.8. Uncheck “Live FFT” in the “Process” pull down menu in Serial EM.
SECTION: Alignment Steps
38.9. Press “Stop” in Camera & Macro Control.    
SECTION: Shut Down Procedure
39. Make sure viewing screen is down. 
SECTION: Shut Down Procedure
40. Make sure condenser and objective apertures are inserted.
SECTION: Shut Down Procedure
41. Magnification is set to SA 9700x.
SECTION: Shut Down Procedure
42. Hit 'Eucentric Focus' and reset the defocus to 0. 
SECTION: Shut Down Procedure
43. Uncheck low dose button in SerialEM to revert the microscope to normal imaging (only for those using low dose operation).
SECTION: Shut Down Procedure
44. Set the spot size to 1.
SECTION: Shut Down Procedure
45. Spread the beam clockwise till the exposure on the FEI set up screen shows 0.60s.
SECTION: Shut Down Procedure
46. Beam/Column Valve is closed (should turn yellow).

SECTION: Shut Down Procedure
47. Filament is turned off. 
SECTION: Shut Down Procedure
48. Go to 'Stage' tab and double click at the center of specimen position to bring the stage to neutral position. 
SECTION: Shut Down Procedure
49. Remove the sample holder by pulling out the rod slowly and then turn clockwise and pull out again. 
SECTION: Shut Down Procedure
50. Insert specimen rob back into the microscope. 
SECTION: Shut Down Procedure
54. Record session time in log book if applicable. 
SECTION: Shut Down Procedure
51. Remove dewar containing liquid nitrogen and dispose appropriately.
SECTION: Shut Down Procedure
52. Go to 'Set Up' tab and find 'Vacuum Supervisor'.
SECTION: Shut Down Procedure
53. At the Cryo tab, press 'Cryo Cycle'.
SECTION: Set up
2. Fill the dewar with liquid nitrogen and cover with foam cap.