Prepare the Cross Grating grid for calibration

Focus the beam and center it (with the trackball).

• Go to microprobe (TEM)
• Push the Eucentric Focus knob on the console.
• In the Direct Alignments window:

1. Adjust the Beam tilt pp X

   • Click on Beam tilt pp X
   • Adjust with Multifunction X/Y knobs
   • Click Done at the bottom of the window.

2. Adjust the Beam tilt pp Y

   • Click on Beam tilt pp Y.
   • Adjust with Multifunction X/Y knobs.
   • Click Done at the bottom of the window.

3. Adjust the Beam shift

   • Click on Beam shift.
   • Adjust with Multifunction X/Y knobs.
   • Click Done at the bottom of the window.

4. Adjust the Rotation center

   • Click on Rotation center.
   • Adjust with Multifunction X/Y knobs.
   • Click Done at the bottom of the window.

Focus the beam and center it (with the trackball).

• Go to nanoprobe (TEM)
• Push the Eucentric Focus knob on the console.
• Check the Condenser 2 centering and adjust if necessary.
• In the Direct Alignments window:

1. Adjust the Beam tilt pp X

   • Click on Beam tilt pp X
   • Adjust with Multifunction X/Y knobs
   • Click Done at the bottom of the window

2. Adjust the Beam tilt pp Y

   • Click on Beam tilt pp Y
   • Adjust with Multifunction X/Y knobs.
   • Click Done at the bottom of the window

3. Adjust the Beam shift

   • Click on Beam shift
   • Adjust with Multifunction X/Y knobs
   • Click Done at the bottom of the window

4. Adjust the Rotation center

   • Click on Rotation center
   • Adjust with Multifunction X/Y knobs
   • Click Done at the bottom of the window

After all calibrations save the settings

• Verify that the Turbo Auto Off Vacuum option is checked (TEM)
• Go to x45k Imaging status
• In the Camera and script window (SEM) open the Setup
• In Record Tab, set 2 sec exposure, processing unnormalized, align frames, save frames and the objective diaphragm out
• If the signal is too weak, increase the time, concentrate the beam or change the carbon area
• Correct astigmatism by CTF

  • Menu: Calibration > Focus & Tuning > Correct astigmatism by CTF
  • Open FFT window with Shift+F
  • Repeat several times until the corrected parameters have converged

• Coma free align by CTF

  • Menu: Calibration > Focus & Tuning > Coma free align by CTF
  • Open FFT window with Shift+F

• Coma vs image shift

  • Menu: Calibration > Focus & Tuning > Coma vs image shift
  • Open FFT window with Shift+F
  • If the signal is too weak, increase the time, set binning, concentrate the beam or change the carbon area
  • parameters for R 1.2/1.3 grids: 5 μm
  • parameters for R 2/2 grids: 6 μm

Introduce the Objective aperture and redo the Correct astigmatism by CTF step once.

Do this configuration just before starting the acquisition on one of the squares selected for the whole acquisition after correction of the image shift in x280 and LD mode imaging states.

• On the square map, mark the positions of the 4 holes at the corners of a 9 holes square.
• Go to the center hole with Go to marker and properly center on the hole with Stage shift.
• Press View in LD mode (ensure that defocus is below -20)
• Open Menu: Navigation > Montaging & grids > Set multi
• Change the parameters of the multishot for calibration

1. Click on the central hole for positioning

• Adjust the number of shots per hole (in general 3)
• Adjust the distance (72 in general for R 2/2 grids)
• Menu: Navigation > Montaging & grids > Find hole in regul to configure

1. Select regular pattern 3 x 3

• ++Calibrate after ensuring proper centering on the center hole|

  • Move by Image shift to the first corner
  • Center properly in the hole using zoom if necessary
  • Save the position by clicking on Save image shift
  • Proceed to the next corner and repeat