LowDose SPA screening

Goal:

• Setup low dose modes.
• View mode should be set up for automated hole centering.
  
• Set camera parameters for all modes.
• We want to cscreen some squares in low dose with on-the-fly frame alignment.
  

SerialEM general settings

• Image Alignment & Focus control panel: check if "center image shift on tilt axis " is de-selected (we do not want unnecessary beam-image shift).
• Image Alignment & Focus control panel: press ResetImageShift to reset any shift to zero.
  .
  

• Low Dose Control: switch on Low Dose Mode, select "continuous update of mag & beam".

Camera settings for all modes

• Camera & Macro Controls, Setup:

• Camera & Macro Controls, Setup, Record mode: check that Dose Fractionation Mode is selected only for Record mode, but do not save frames:
  
  

• For frame alignment parameters, select parameters 'SPA screening', set "In SerialEM plugin":
  
  

Record mode setup

• WITH THE SCREEN DOWN, Low Dose Control: "Area to show when screen down" select Record and adjust the beam and magnification with the Titan hand panels.
• We mostly use Nanoprobe spot 6 or 7, aim for a camera dose rate of ~15 e/pixel/sec with the sample in the beam, the objective aperture inserted and the energy slit inserted.
• If necessary, center beam in Titan UI with Direct Alignment/Tomo Beam shift.
• Go to an empty area.
• Take a Preview exposure.
• SerialEM menu Calibration, Electron Dose:
       

Focus/Trial/Preview mode setup

• Low Dose Control: while in Record mode, click on T and F in "Copy current area mag & beam to".

View mode setup

• For View mode set a magnification that will cover just one entire hole, same spot size as used for Record/Focus/Trial/Preview, Offsets for View -100 micron defocus.
• If beam centering is off in View mode: in Low Dose Control, Additional Beam Shift, select Set, center beam with the left trackball, de-select Set. This stores an additional beam shift for just the View mode.
• Low Dose Control: de-select "continuous update of mag & beam".

Focus/Trial distance and angle

• Take a View image and position the Focus/Trial area conveniently on some carbon area by changing the Focus/Trial distance and the Rotate inter-area axis.
  
  

• Camera & Macro Controls: take a View, Trial, Focus and Preview image, twice in this order.
  

• If the beam is off in Focus/Trial/Record/Preview: center it in any of these modes with Direct Alignment/Tomo Beam Shift.
• If the beam is off in View: Low Dose Control, Additional Beam Shift, select Set, center beam with the left trackball, de-select Set.

• Insert and center an objective aperture in Record mode.
• Set a desired autofocus target, e.g. -3.5 um in SerialEM Menu Focus/Tune, Set Target.
• Go to some squares.
• Center a hole with View.
• AutoFocus.
• Record image (takes a bit of time).
• Add markers in the Navigator to keep track of good squares.