tSEM Imaging

Owned by
Masaaki Kuwajima
Last updated: Aug 13, 2021
2 min read

Basic workflow for serial section imaging

Status check

Specimen exchange

Establish vacuum

Insert STEM detector

Turn on EHT

Beam alignment

Navigate to ROI(s)

Store ROIs with ATLAS

Execute automated imaging with ATLAS

Check image quality


Before you start your work, check SEM status

  • Vacuum: System Vac should be in 10-6 range; Gun vac should be in 10-10 range
  • Gun status: EHT should be off; Gun should be on; If you don't see the EHT on option in the drop-down menu, you may need to restart the instrument.
  • Current/Voltage
  • Location of STEM detector
  • Location of the stage - "Safe STEM"


Exchanging grids: 

To remove grids from the SEM

  1. Turn the EHT off
  2. Double click on 'Safe STEM'
  3. Click 'STEM out'
  4. Turn on the N2 gas
  5. Under the Vacuum tab, click on 'Vent'
  6. Wait for the chamber to completely vent, then open the chamber door
  7. Turn off the N2 gas
  8. Be sure to wear gloves before removing grid holder. Do not touch the grid holder or anything inside the chamber without wearing gloves
  9. Exchange grids and be sure to document what grids were removed and what grids were added

To add grids to the SEM

  1. Load grid holder into chamber and close the door

  2. Under the Vacuum tab, click on 'Pump'

  3. Double click 'Safe STEM'

  4. Once there is a green check mark next to Vacuum, click 'Stem In'

  5. Double click on a position in the Stage Point List

  6. Check vacuum levels to see if they are steady. If so, turn EHT on


Four routine gun alignment checks:

Gun alignment

  1. Scroll to an area on the aluminum flat surface of the grid holder. Set detection mode to SE2. Focus the image

  2. Under the Aperture tab, click on 'gun alignment'

  3. Click 'Emission'

  4. Set scan speed to 9

  5. Go back to the aperture tab and center beam if necessary by using the sliders on the Gun Alignment grid

Aperture alignment

  1. Scroll to a tissue section on the grid

  2. Click on STEM and then click 'Normal' 

  3. Click on aperture alignment

  4. Click  'focus wobble on' in the SmartSEM Status window

  5. Adjust alignment accordingly with the aperture x and y knobs on the keyboard

  6. Click 'focus wobble off' in the SmartSEM Status window

Objective current wobble

  1. Click 'objective current wobble on' in the SmartSEM Status window

  2. Adjust wobble with aperture x and y knobs accordingly

  3. Click 'objective current wobble off' 

Stigmation calibration

  1. Click and expand side bar
  2. Click on calibrate stigmation
  3. Adjust stigmation with the stigmator knobs accordingly
  4. Adjust stigmation further with the calibration window if necessary


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