BECM

Pivot Points Alignment

Pivot Points Alignment

This alignment should be used before setting up a new acquisition using a grating grid with latex beads. This alignment is very important for a subsequent collection which is using a beam tilts (Image shift).

Do not perform this directly after refilling LN2 (ideally > 3 hours), and not directly after using LOW MAG mode.

Pivot Tilt

Beam tilt pivot points

  1. Go to standard focus by double clicking “STD Focus” (right hand panel).
  1. Ensure that you are at eucentric height. Note: Use rough eucentric for a rough targeting. Measure the actual defocus in SerialEM (Menu –> Focus/Tune –> Measure defocus). The log file will display the actual defocus. Use the Z up and Z down buttons (small panel) to adjust the eucentric height. and confirm/readjust with “Measure Defocus”
  1. Select record beam conditions in SerialEM (pink panel, Rec.)
Figure 1: Center the beam

 

  1. Condense the beam to a small point (Brightness dial, left hand panel) and roughly center the beam on the large phosphor screen with “Shift X and Y”(both hand panels) (Figure 1).
  1. Insert small phosphor screen “FS retract” right hand panel.
Figure 2: Center beam on Focus screen

 

  1. Condense the beam further into a very small dot (Brightness dial, left hand panel) and center the beam on this screen with “Shift X and Y” (both hand panels) (Figure 2).
Figure 3: Wobbler window

 

  1. Open the wobbler window in TEM center through the “Lens/Deflector” window by hovering over “Wobbler” and clicking on “Wobbler window” (Figure 3).
Figure 4: Tilt X,Y, wobbler window

 

  1. Select in the new window “Tilt X” (Figure 4). CL Comp tilt will be automatically assigned to the Def/Stig knobs.
  2. Use “Def/Stig X” knob (left hand panel) and minimize the displacement of the major beam. Note: You can use Beam shift if you will lose the beam from the small Phosphor Screen.
  3. Switch off “Tilt X” and switch on “Tilt Y”.
  4. Use “Def/Stig Y” knob (left hand panel) and minimize the displacement of the major beam. Note: You can use Beam shift if you will lose the beam from the small Phosphor Screen.
  5. Switch off “Tilt Y”.

Beam Tilt

As above you still need to be at standard focus and eucentric height.

  1. Remove the Small Focus Screen (“FS insert”, right hand panel)
  1. Put the beam in parallel mode by clicking “Parallel” in the microscope pictogram. It will be highlighted green.
Figure 5 Grating grid with the latex beads

 

  1. Center the image with the phosphor screen using the stage on one of the latex beads, center the beam (Figure 5).
  1. Insert the small focus screen “FScreen retract”, (right hand panel).
  1. Center the latex bead on the screen using the trackball.
  1. Paint a circle around the position of the latex bead (TEM Center menu –> Tools –> Ellipse)
  1. Change the defocus to -10 to -15 um (“Image/diff focus button”, right hand panel).
  1. Switch on “Bright tilt” (Left hand panel). This will assign BeamAlign2 to the Def/Stig knobs.
  1. Center the latex bead again on its original position using “Def/Stig X&Y” buttons (both hand panels).
  1. Hit standard focus (Std focus, right hand panel).
  1. Repeat above procedure until the bead does not change its position during defocusing.
  1. Switch off “Bright tilt” (Left hand panel).

Pivot Shift

Beam shift pivot points

From before you should still be at standard focus and eucentric height. You should also still have a latex bead in the center of the focus screen

  1. Remove condenser lens aperture (CL button, microscope pictogram, TEM center).
  2. Set defocus to . -40 um (“Image/Diff focus” button, right hand panel).
  1. In the Lens/Deflector window hover over “CL Comp Shift” and push “assign to knobs”.
  1. In SerialEM edit the script Shift X,Y (menu –> Script –> Edit –> Edit 6 (or similar)) and put amp_X to 40 and amp_Y to 0, then run script Shift X,Y (Figure 8).
Figure: 8 Shift XY script

 

  1. Minimize the movement of the latex bead along the beam motion on the focusing screen using “Def/Stig X”button.
  2. If you see lateral motion against the beam motion, assign “CL Comp Angle” to the knobs (via Lens/Deflector window –> CL Comp Angle –> Assign to knobs”.
  3. Click on one of the “Def/Stig” button to go into coarse mode.
  4. Use the “Def/Stig X”button to minimize the lateral motion against the beam shift.
  5. Click on one of the “Def/Stig” button to go back into fine mode.
  6. Stop script execution in Serial EM (dark green panel, “STOP”)
  7. Set BEAM ALIGN 1 to default by hovering in the LEns/Deflector window over Beam Align 1 and hit “Default”.
  8. Edit script Shift X,Y and put amp_X to 0 and amp_Y to 40, then run script Shift X,Y (Figure 8).
  1. Minimize the movement of the latex bead along the beam motion on the focusing screen using “Def/Stig Y”button.
  2. If you see lateral motion against the beam motion, assign “CL Comp Angle” to the knobs (via Lens/Deflector window –> CL Comp Angle –> Assign to knobs”.
  3. Click on one of the “Def/Stig” button to go into coarse mode.
  4. Use the “Def/Stig Y”button to minimize the lateral motion against the beam shift.
  5. Click on one of the “Def/Stig” button to go back into fine mode.
  6. Stop script execution in Serial EM (dark green panel, “STOP”)
  7. Unassign “CL Comp Angle” from the knobs (via Lens/Deflector window –> CL Comp Angle –> Assign to knobs”). Button will not be green anymore.
  1. Set BEAM ALIGN 1 to default by hovering in the LEns/Deflector window over Beam Align 1 and hit “Default”.
  2. Insert condenser lens aperture (CL button, microscope pictogram, TEM center).
  3. Push STANDARD FOCUS (“STD focus”, right hand panel).
  4. Center the beam with “Shift X&Y” button (both hand panels).