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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 and stage - Safe stem
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
Exchanging grids:
To remove grids from the SEM
- Turn the EHT off
- Double click on 'Safe Stem'
- Click 'Stem out'
- Under the Vacuum tab, click on 'Vent'
- Turn on the N2 gas
- Wait for the chamber to completely vent, then open the chamber door
- Turn off the N2 gas
- Remove grid holder and be sure to wear gloves. Do not touch the grid holder or anything inside the chamber without wearing gloves
- Exchange grids and be sure to document what grids were removed and what grids were added
To add grids to the SEM
Load grids into chamber and close the door
Under the Vacuum tab, click on 'Pump'
Double click 'Safe Stem'
Once there is a green check mark next to Vacuum, click 'Stem In'
Double click on a position in the Stage Point List
Check vacuum levels to see if they are steady. If so, turn EHT on
Four routine gun alignment checks:
Gun alignment
Scroll to an area on the aluminum flat surface of the grid holder. Set detection mode to SE2. Focus the image
Under the Aperture tab, click on 'gun alignment'
Click 'Emission'
Set scan speed to 9
Go back to the aperture tab and center beam if necessary by using the sliders on the Gun Alignment grid
Aperture alignment
Scroll to a tissue section on the grid
Click on STEM and then click 'Normal'
Click on aperture alignment
Click 'focus wobble on' in the SmartSEM Status window
Adjust alignment accordingly with the aperture x and y knobs on the keyboard
Click 'focus wobble off' in the SmartSEM Status window
Objective current wobble
Click 'objective current wobble on' in the SmartSEM Status window
Adjust wobble with aperture x and y knobs accordingly
Click 'objective current wobble off'
Stigmation calibration
- Click and expand side bar
- Click on calibrate stigmation
- Adjust stigmation with the stigmator knobs accordingly
- Adjust stigmation further with the calibration window if necessary