• Introduce the Cross Grating Grid in the column.
• Go to x34 Imaging status.
• Select 2 positions: a carbon square and an empty square.
• Introduce the Condenser 2 of 30 um
• Go to x280 Imaging status.
• In DigitalMicrograph click View and place the mouse arrow on a feature.
• Use Alpha Toogle 20° in Stage² window (TEM) to rock the grid.
• Correct with Z axis knobs on the console.
• Repeat a second time.
• Go to x45k Imaging status
• In Low Dose Control window (SEM) click on Go to: Record.
• Push the Eucentric Focus knob on the console.
• Inset the screen (FC).
• Push the Wobbler knob on the console.
• Adjust the Z height on the screen with the Z axis knobs on the console.

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