Practical Electron Microscopy and Database

• Sign the log sheet and inspect the microscope for any issues before starting (ensure the chiller temperature is around 70, water flow readings are: DP48, OL38, CL20, Gate60, and SIP pressure is approximately 5.8 x 10⁻⁵ Pa, with the column pressure around 5x10⁻⁴).
• Refill the anti-contamination device (ACD) tank with liquid nitrogen (LN2) to minimize sample contamination from the electron beam. Be sure to wear the necessary protective gear while handling LN2..

  

• Electron Beam Generation
  • Ensure that all apertures are removed from the electron beam path.
  • Activate the electron beam by selecting "Filament ON.": In the High Voltage Control window, click "Filament ON" or press the Beam Switch on the left Control Panel to generate the electron beam. (If the beam current does not register any change after turning the filament on, the filament might be burnt out.)
  • If the electron beam is visible, proceed to aligning the TEM system. If not, switch to TEM mode, set the Spot size to 2, α = 3, and choose MAG2 on the right Control Panel.
  • Use the BRIGHTNESS knob to check for the appearance of the electron beam on the fluorescent screen. Adjust both the BRIGHTNESS and SHIFT X/Y knobs to make the beam visible and bright. If the beam does not appear, try moving the specimen or grid to see if they are obstructing it. If you're still unable to find the beam, seek assistance.

• Align Condenser Lens (CL) Aperture.
  • Set the microscope to MAG1 and choose an appropriate magnification, for example, 40kx.
  • Insert the largest condenser aperture, with CL Aperture knobs shown below, into the column, and center it as follows:

    

    • Use the BRIGHTNESS knob to create the smallest possible electron beam, then center the beam using the SHIFT X and Y knobs.
    • Gradually expand the electron beam by adjusting the BRIGHTNESS knob. If the beam shifts away from the center of the screen as it expands, recenter it using the aperture adjustment knobs.
    • Adjust the aperture knobs so that the beam expands and contracts symmetrically around the center when you turn the BRIGHTNESS knob back and forth near the focus position.
    • Insert the desired CL aperture (options include 150 µm, 70 µm, 50 µm, and 10 µm) and center it in the same way.
• Correct any Condenser Lens (CL) astigmatism.
  • Astigmatism in the CL will cause the focused beam to appear elongated on the screen. To fix this, adjust the CL lens so that the beam forms a perfectly round spot.
  • Use the BRIGHTNESS knob to focus the electron beam.
  • Press the "COND STIG" button to enter astigmatism correction mode.

    

  • Slowly rotate the BRIGHTNESS knob back and forth near the focus point, then use the DEF/STIG X and Y knobs to adjust the beam's shape until it becomes round, both slightly before and after the focus position.
  • Press the "COND STIG" button again to exit the correction mode.
• Set the eucentric height by pressing the "STD Focus" button and adjusting the Z buttons to focus the image:
  • Find the specimen or carbon network on the grid, and set the magnification to 40Kx.
  • Press the "STD FOCUS" button on the right Control Panel.
  • Press the "IMAGE WOBB X" button.

    

  • Use the Z-height controls to adjust the focus until the image has minimal contrast.

    

  • Once focused, press the "IMAGE WOBB" button again to deselect it.
  • After completing this alignment, perform all coarse focusing using the Z-height adjustment.
• Perform gun shift correction at spot size 1 and condenser shift correction at spot size 5, repeating as necessary:

③ in the figure above is for adjusting Spot Size. The arrows change the beam spot size. Each time you click on the right arrow button, the spot size (minimized using the BRIGHTNESS control) increases by one step. Every time you click on the left arrow, the spot size decreases by one step.

• Switch to MAG mode and set the magnification to 40kx.
• Perform gun shift correction at spot size 1: Set the SPOT SIZE knob to 1 and focus the electron beam using the BRIGHTNESS knob.
• In the Alignment Panel for Maintenance, click "DEF Select Gun."

  

• Use the SHIFT X and Y knobs to center the beam on the fluorescent screen.
• Once centered, click "DEF Select Gun" to exit the mode.
• Perform condenser shift correction at spot size 5: Set the SPOT SIZE knob to 5 and focus the electron beam using the BRIGHTNESS knob.
• Press the "BRIGHTNESS TILT" switch or click "DEF Select CLA" in the Alignment Panel for Maintenance.

  

• Center the electron beam on the screen using the SHIFT X and Y knobs.
• Deselect "DEF Select CLA."
• Repeat the two steps above until the electron beam remains centered on the screen when you change the SPOT SIZE.

• Adjust condenser lens (CL) deflection coils by adjusting Tilt X and Y and shift X and Y (figure below). This adjustment consists of adjusting the ratio of the currents in the 1st and 2nd CL deflection coils so that the electron beam spot remains stationary when you tilt the electron beam.

  

  • Focus electron beam using the BRIGHTNESS knob.
  • Press BRIGHT TILT and center the beam using the SHIFT X and Y knobs.

    

  • Press BRIGHT TILT to deselect it when done.
  • Click Compensator Tilt in the Alignment Panel for Maintenance window.

  

• Align the voltage axis by clicking "Wobbler HT." The image should expand and contract symmetrically around the center. If not, click the "BRIGHT TILT" button and adjust DEF/STIG until the image behaves correctly..

  Wobbler HT	BRIGHT TILT

• Correct for objective lens astigmatism.

Tilt X and Y adjustment:
        Tilt adjustments in the X and Y directions involve modifying the ratio of currents in the upper and lower CL deflection coils to ensure the electron beam spot remains stationary during tilting.

• Open the TEM Controller menu, select Maintenance, and click Alignment to display the Alignment Panel for Maintenance. Set the magnification to 40,000× which uses the ray pass shown in an image below.

  TEM Controller	Maintenance > Alignment

  

• Use the BRIGHTNESS knob to focus the electron beam. This knob is ued to converge and spread the electron beam.
• Press BRIGHT TILT or click DEF Select CLA in the Alignment Panel for Maintenance.

  BRIGHT TILT	DEF Select CLA

• Center the beam with the SHIFT knobs.
• Click Compensator Tilt in the Alignment Panel for Maintenance.

  

• Press the Wobbler-Tilt X button in the Alignment Panel (see the image above).
  • If the current ratio in the upper and lower deflection coils is incorrect, the beam spot will split.

• Use the DEF/STIG X knob to merge the split spot.
  • If the spot moves off the screen, reduce the magnification and continue adjusting.
  • If the beam splits in the Y direction, click Compensator Angle in the Alignment Panel (see the image below) and use the DEF/STIG X knob to unify the spot. Then, turn off Compensator Angle.

    

• Turn off Wobbler-Tilt X in the Alignment Panel.

• If the beam drifts from the screen center when adjusting BRIGHTNESS, re-center it using the SHIFT X/Y knobs (see the image below) after pressing BRIGHT TILT or clicking DEF Select CLA.

  BRIGHT TILT	DEF Select CLA

• Press the Wobbler-Tilt Y button in the Alignment Panel. The beam spot will split in the Y direction.

  

• Use the DEF/STIG Y knob (see the image below) to merge the split spot.

  

  • If the spot moves off the screen, lower the magnification and continue adjustments.
  • If the spot splits in the X direction, click Compensator Angle in the Alignment Panel (see the image below) and use the DEF/STIG Y knob (see the image below) to unify it. Turn off Compensator Angle once the spot is unified.

    

• If the beam drifts from the center when adjusting BRIGHTNESS, re-center it using the SHIFT X/Y knobs (see the image below) after pressing BRIGHT TILT or selecting DEF Select CLA.

• Finally, click the Compensator Tilt button to turn it off.

Shift X and Y adjustment:
       The Shift X and Y adjustments involve modifying the ratio of currents in the upper and lower CL deflection coils to ensure the caustic spot tilt remains stable when the spot is shifted.

• From the TEM Controller menu bar, select Maintenance and click Alignment to open the Alignment Panel for Maintenance window. Press the SA DIFF switch (R1-②) to enter DIFF mode.

  

• Rotate the BRIGHTNESS knob (L1-⑥) fully counterclockwise.

• Use the DIFF FOCUS knob (R1-⑦) to focus the caustic spot (see the image below) on the fluorescent screen.

  

• Click on Compensator Shift.

• Click on Wobbler Shift X. If the current ratio in the upper and lower deflection coils is not correctly adjusted, the caustic spot will split into two parts along the X-axis.

• Use the DEF/STIG X knob (L1-⑩) to unify the split spot. If the caustic spot moves off the screen, reduce the camera length and continue adjusting. If the electron spot splits in the Y direction, click on Compensator Angle in the Alignment Panel for Maintenance (see Fig. 5.26), then unify the spot using the DEF/STIG X knob (L1-⑩). After ensuring the spot is unified, turn off Compensator Angle.

• Click on Wobbler Shift X to disable it.

• If the caustic spot moves from the screen center when adjusting the BRIGHTNESS knob, use the DEF/STIG X and Y knobs (L1-⑩, R1-④) to center the beam after pressing BRIGHT TILT (L1-⑤) or selecting DEF Select CLA in the Alignment Panel for Maintenance.

• Click on Wobbler Shift Y. If the current ratio in the upper and lower deflection coils is not correctly adjusted, the caustic spot will split into two parts along the Y-axis.

• Use the DEF/STIG Y knob (R1-④) to unify the split spot. If the caustic spot moves off-screen, shorten the camera length and continue the adjustment. If the electron spot splits in the X direction, click on Compensator Angle in the Alignment Panel for Maintenance (see Fig. 5.26), then unify the spot using the DEF/STIG Y knob (R1-④). Once unified, turn off Compensator Angle.

• Click on Wobbler Shift Y to disable it.

• Turn off Compensator Shift Y.

• If the caustic spot shifts off-center when adjusting the BRIGHTNESS knob, use the DEF/STIG X and Y knobs (L1-⑩, R1-④) to re-center the caustic spot after pressing BRIGHT TILT (L1-⑤) or selecting DEF Select CLA in the Alignment Panel for Maintenance.

Centering the Voltage Axis:

        The goal is to adjust the TEM optics so that the image remains centered on the fluorescent screen even when you change the accelerating voltage.

• Press the "MAG1" switch to obtain an image.

• Use the OBJ FOCUS knob to focus the image.

• Expand the beam across the entire screen and center a prominent feature of the image.

• Press the "HT WOBB" switch. The image will expand and contract periodically.

• Press the "BRIGHT TILT" button.

• Adjust the DEF/STIG controls until the image expands and contracts symmetrically around the center of the screen.

• Press the "HT WOBB" button again to stop the wobbling and deselect the "BRIGHT TILT" button.

Objective Lens (OL) Astigmatism Correction:
      To achieve high-quality imaging, it is important to correct any OL astigmatism, as this can distort the image quality. Perforated amorphous films like a carbon grid are ideal for testing and correcting OL astigmatism.

• Locate an amorphous area on the carbon grid and position it at the center of the screen.

• In MAG1 mode, set the magnification to 100kx or higher.

• Raise the screen.

• Insert the CCD camera and click "Start View" in the Digital Micrograph window to display the image of the amorphous material.

• In the Digital Micrograph window, navigate to Process -> Live -> FFT.

• A well-corrected OL astigmatism will produce a spherical FFT (Fast Fourier Transform).

• Press the "OBJ STIG" button to enter astigmatism correction mode.

• Adjust the DEF/STIG controls to further improve the FFT sphere.

• Press the "OBJ STIG" button again to exit the mode.

Control Panel L1:

• ① BEAM Switch: Pressing this switch starts emitting electron beams when the built-in lamp is off. If the lamp is on, pressing the switch stops the electron beams. This function is identical to the Filament ON/OFF switch in the High Voltage Control window.
• ② APERTURE CONTROL: Pressing one of the motor-driven aperture switches (CL, EDS, OL, HC, or SA) illuminates the built-in lamp. The aperture-selector switches (OPEN, 1, 2, 3, and 4) and the X/Y arrow switches then control the selected motor-driven aperture. Note: Apertures cannot be selected if they are not motor-driven.
• Aperture switches:
  • CL: Selects the condenser aperture.
  • EDS: Selects the hard X-ray aperture.
  • OL: Selects the objective aperture.
  • HC: Selects the high-contrast objective aperture.
  • SA: Selects the field-limiting aperture.
• Aperture selector switches:
  • OPEN: Opens the aperture.
  • 1, 2, 3, 4: Selects aperture size number for the chosen aperture assembly.
• X and Y arrow switches: Move the aperture in the X and Y directions.
• CRS (Coarse) switch: When activated, the aperture moves 16 times further in the X or Y directions with each press.

• ③ PROBE CONTROL:
  • Illumination mode selector switches (TEM, EDS, NBD, CBD):
    • TEM switch: Lights the lamp and sets the illumination mode to TEM (wide-area illumination).
    • EDS switch: Lights the lamp and sets the illumination mode to EDS (high-current density micro-area illumination).
    • NBD switch: Lights the lamp and sets the illumination mode to NBD (small convergence angle micro-area illumination).
    • CBD switch: Lights the lamp and sets the illumination mode to CBD (changeable convergence angle micro-area illumination).
  • α SELECTOR knob: Adjusts the convergence angle without changing the illuminating spot size. Turning the knob clockwise decreases the CM lens current and increases the illuminating angle. Turning the knob fully clockwise decreases the CM (condenser minilens) lens current to zero.
  • SPOT SIZE knob: Adjusts the spot size of the electron beam when converged to minimum size using the BRIGHTNESS knob. Increasing the SPOT SIZE number decreases the spot size, while decreasing the number increases the spot size.

• ④ ROOM LAMP Switch: Turns the room lamp on or off when provided.

• ⑤ DEF/STIG Switches: Pressing any of these switches lights the built-in lamp and allows control of the coil current through the SHIFT or DEF/STIG knobs. Pressing the switch again turns off the lamp and maintains the current coil setting.
  • IMAGE SHIFT switch: Shifts the image at high magnifications.
  • PLA switch: Adjusts the projector lens deflector coil current for shifting diffraction spots or specimen images.
  • COND STIG switch: Adjusts the condenser lens stigmator coil current during astigmatism correction.
  • OBJ STIG switch: Loads the objective lens stigmator coil’s current from memory and sets the DEF/STIG knobs for astigmatism correction.
  • DARK TILT switch: Adjusts the beam deflector coil current for dark-field imaging by loading a preset from memory.
  • BRIGHT TILT switch: Adjusts the beam deflector coil current for bright-field imaging by loading a preset from memory.
  • NTRL switch: Clears current data from memory and brightens the built-in lamp.

• ⑥ and ⑦ BRIGHTNESS:
  • ⑥ BRIGHTNESS Knob: Adjusts the electron beam’s convergence and spread.
  • ⑦ BRIGHTNESS CRS Switch: Increases the current change per notch of the BRIGHTNESS knob by 16 times when activated.

• ⑧ and ⑨ SHIFT:
  • ⑧ SHIFT X Knob: Shifts the electron beam in the X direction by adjusting the condenser lens beam deflector coil current. If DEF Select – Gun is chosen in the Maintenance window, the electron gun 1st beam deflector coil current is adjustable.

    

  • ⑨ SHIFT CRS Switch: Increases the current change per notch of the SHIFT knobs by 16 times when activated.

• ⑩ and ⑪ DEF/STIG:
  • ⑩ DEF/STIG X Knob: Controls the X current in the selected coil (as determined by the DEF/STIG switch or the Maintenance window settings).
  • DEF/STIG CRS Switch: Increases the current change per notch of the DEF/STIG knobs by 16 times when activated.

Control Panel R1:

• ① Wobbler Switches:
  • IMAGE WOBB X: Used for focusing. While this switch is on, the current in the 1st and 2nd beam deflector coils fluctuates periodically. If the image is out of focus, it will wobble in the X direction. This function is equivalent to the ImageX button in the Alignment Panel for Maintenance window.
  • IMAGE WOBB Y: Used for focusing. While this switch is on, the current in the 1st and 2nd beam deflector coils fluctuates periodically. If the image is out of focus, it will wobble in the Y direction. This function is equivalent to the ImageY button in the Alignment Panel for Maintenance window.
  • HT WOBB: While this switch is on, the high voltage fluctuates periodically, aiding in voltage axis alignment. This function is equivalent to the HT button in the Alignment Panel for Maintenance window.

    

• ② Function Switches:
  Used to select an image-forming mode. The magnification or camera length in the chosen mode can be adjusted using the MAG-CAM L knob and is shown on the monitor. The set magnification or camera length is saved, so it can be restored by selecting the original mode again, except for MAG 2 mode.
  • MAG 1 switch: Selects the normal magnification mode.
  • MAG 2 switch: Switches to a specific magnification that is not stored.
  • LOW MAG switch: Selects the low magnification mode.
  • SA MAG switch: Selects the selected-area magnification mode.
  • SA DIFF switch: Selects the selected-area diffraction mode.

• ③ SHIFT Y Knob. Moves the electron beam in the Y direction by adjusting the condenser lens beam deflector coil current. The 1st electron gun beam deflector coil current adjusts when DEF Select – Gun is chosen from the Alignment Panel for Maintenance window.

  

• ④ DEF-STIG Y Knob: Adjusts the Y current in the coil selected by the DEF/STIG switch or DEF Select buttons in the Alignment Panel for Maintenance window.

• ⑤ MAG/CAM L Knob: Changes the normal magnification when the MAG 1 or MAG 2 switch is selected. When the LOW MAG switch is selected, it adjusts the low magnification. It also adjusts the selected area magnification in SA MAG mode or the camera length in SA DIFF mode. Turning it clockwise increases the magnification or camera length, and turning it counterclockwise decreases them.

• ⑥ OBJ FOCUS Knob: Focuses the image by adjusting the objective lens current (objective minilens current in LOW MAG mode).
  • FINE knob: Provides the smallest current change per notch.
  • COARSE knob: Provides 16 times the current change per notch compared to the FINE knob.
  • CRS Switch: When activated, increases the current change per notch of the FINE and COARSE knobs by 16 times.

• ⑦ DIFF FOCUS Knob: Focuses the field-limiting aperture when the SA MAG switch is on and focuses the diffraction pattern when the SA DIFF switch is on.

  

  • CRS Switch: Multiplies the current change per notch of the FOCUS knob by 16 when activated.

• ⑧ EXP TIME/PHOTO:
  • EXP TIME knob: Adjusts the exposure time. Turning it clockwise increases the exposure time, and counterclockwise decreases it.
  • AUTO switch: Activates the automatic exposure mode, illuminating the built-in lamp. Pressing it again switches to manual exposure mode, turning off the lamp.
  • PHOTO switch: Advances the film to the exposure position when pressed (if the lamp is unlit). When the film reaches the position, the lamp lights, and the film is exposed and then advanced. Pressing the switch again (when the lamp is lit) exposes the film. Film advancement and exposure timing can be set in the Film Camera Property window. Lamp indicates that the shutter is open when lit.

• ⑨ STD FOCUS Switch: Resets the objective lens current to its original reference value.

• ⑩ F Switches: Allows the operator to assign specific functions to the F1 to F6 switches.

• ⑪ Z Switches: Moves the specimen vertically.