High Resolution Microscopy

High Resolution Microscopy

& Advanced Imaging

TEM startup and alignment

Transmission Electron Microscope
Revised 8/28/2006

1. Starting up the Instrument:

  1. Turn on nitrogen tank (open main valve on top all the way) and adjust to 3-4 lbs/square inch with the front valve (large “T” shaped valve). Reading may be high until the TEM is turned on for a while. Leave the nitrogen on while the TEM is running.
  2. Turn the TEM on. Turn key switch to 50kV or 80kV (50 kV is used for most biological applications); check that the magnification is set at 3000x.
  3. Turn on cooling unit behind TEM by pushing the green “start” button.
  4. Wait for the TEM to pump down. A vacuum is created and the pressure must decrease to at least 2 x 10-5 in order to operate the scope. When the pressure is low enough, the “open V3” light will illuminate on the left panel, and the green “open V3” light will illuminate on the separation valve (located on right side of column).
  5. Open the V3 aperture (separation valve on right side of column).
  6. Press and hold the µA button (on right panel), and then, while still holding in this button, press the green BEAM button on the left panel. Look for a brief deflection on the voltage meter (right panel), which indicates high voltage.
  7. Press the FIL button (on left next to the BEAM button) to activate the filament.

2. Beam and Filament Alignment and Stigmation:

  1. Bring to crossover (when the beam is at its most focused point). The rectangular screen should be flipped down.
    • Turn the condenser knob (small, outermost knob next to the word “brightness” counter clockwise so the beam is at its smallest diameter.
  2. Align the beam at its optical center with the small, black alignment knobs on the right panel. The optical center is halfway between the center (black dot) and top edge of the small screen.
  3. Under saturate the filament by turning the filament knob counter clockwise until a halo appears.
  4. Center the halo within the beam (so it is as symmetrical as possible around the inner circle) with the filament pre-centering knobs.
  5. Adjust for astigmatism with the C2 stigmation knobs (right panel). Adjust so there is no distortion of the shape of the filament image in the center of the halo when the beam is brought to crossover and back with the condenser knob.
  6. Slowly turn the filament knob clockwise until the halo just disappears-just past saturation. The beam should look bright green.
  7. Spread the beam by turning the condenser knob clockwise. The beam should spread concentrically, and then return to the optical center when brought back to crossover.
  8. If the beam does not spread concentrically, the condenser and contrast apertures must be adjusted
    • Adjust the beam with the condenser knob so that it takes up approximately half of the circular screen.
    • Slowly turn the knobs on the condenser aperture (top left on column) until the partially spread beam is centered.
    • The contrast aperture (right-middle of column) may also need to be aligned in the same way to allow even spreading of the beam.  Both apertures must be centered and aligned with each other for the beam to spread evenly.
    • Fully spread the beam and check that it reaches the outer edge of the screen evenly. Additional slight adjustment of the condenser and contrast apertures may be needed.

3. Objective Stigmation:

  1. Insert a “holy” grid into the specimen chamber and focus it first at 3000x magnification.
  2. Find a very small hole, as circular and symmetrical as possible.
  3. Slowly increase the magnification, centering and focusing on the small hole with each increase.
  4. Increase the magnification until it is at lease twice that which you will view your samples at (if the hole looks too large or misshapen at high magnification, find a different one).
  5. Using the course and fine focus knobs, get the hole into clear focus.
  6.  Now slowly turn the fine focus knob clockwise until a “halo” is clearly visible around the hole. The halo will appear as a dark concentric line surrounded by a light concentric line, just inside the margin of the hole.
  7. Carefully adjust the OBJECTIVE STIG knobs (left side) until the halo is symmetrical around the hole. At this point the dark interior line will appear even all the way around. If the stigmatism is off, this line will appear blurred in some parts. Adjust so it is clear and even around the hole.
  8. Now slowly turn the fine focus knob COUNTERCLOCKWISE so the halo becomes as small as possible without disappearing. Carefully adjust the objective stigmation so it is still symmetrical.
  9. Remove and carefully store the holy grid.

4. Projector Stigmation: (check and adjust each time you change specimens)

  1.  Turn the condenser far clockwise.
  2. Set the mode switch (on right) to “D”.
  3. Adjust the image so it looks like a 3-legged star. Use the small, outer knob on the fine focus and the condenser to properly view the star. Then use the P1 stigmator knobs to adjust to the 3-legged star (the 4-legged star will become very small and will eventually turn into the 3-legged star).
  4. Switch back to HR mode and insert the specimen.

5. Introducing a Specimen

Before introducing a specimen:

Fill anti-contamination device (canister on left side of column) with liquid nitrogen and cover. Wait 5 minutes, then top off the canister with liquid nitrogen. You may now introduce and view the specimen. Check the liquid nitrogen level after about 1 hour and refill. The refill should last all day.

Check and Adjust Stigmation and Alignment as Necessary (ask for assistance if you have never done this before)

  • Open the V3 separation valve if it is closed (be sure the green light on the valve is illuminated first), then turn on the beam and filament:
    • Turn the condenser knob far to the right. Press and hold the µA button (on right panel), and then, while still holding in this button, press the green BEAM button on the left panel. Look for a brief deflection on the meter (right panel), which indicates high voltage.
    • Press the FIL button (on left next to the BEAM button) to activate the filament.
  • Check the condenser and projector stigmation and adjust as necessary. Check that the beam spreads concentrically and adjust the condenser and contrast apertures if necessary (see “Startup and Alignment).
  • Objective stigmation may be checked and adjusted at this time as well, using the “holey grid”. Do not attempt this adjustment without proper training.
  • Turn off beam and filament before introducing specimen (reason: if the pressure happens to rise above 2-5, high voltage may damage the specimen).

Introducing and Viewing the Specimen:

  • Pull the sliding bar of the specimen chamber all the way out (and in 12 o’clock position).
  • Turn the airlock counterclockwise until the specimen cartridge is visible (6 o’clock position).
  • Screw the threaded end of the specimen holder tool into the specimen cartridge and carefully remove it from the chamber.
  • Close the airlock by turning it clockwise to the 12 o’clock position.
  • Place the specimen cartridge on the holder and attach the tool to the end that holds the grid.
  • Elevate the head to a vertical position, taking care not to touch it with your hands.
  • Carefully unscrew the grid holder by turning it counterclockwise.
  • Place the specimen grid (specimen-side up) in the grid holder and screw the cap back on.
  • Slowly lower the head and then remove the tool.
  • Open the airlock and slide the specimen holder into the chamber.
  • Close the airlock, and then press the green LOCK button on the left panel (the green “lock” light should be illuminated.
  • Immediately after pushing the lock button, slowly move the sliding bar to the 3 o’clock position and wait for two loud clicks.
  • After the clicks, wait 10 seconds, then push the sliding bar down to the 6:00 position and then all the way forward.
  • Turn on the beam and filament:
    • Turn the condenser knob far to the right. Press and hold the mA button (on right panel), and then, while still holding in this button, press the green BEAM button on the left panel. Look for a brief deflection on the voltage meter (right panel), which indicates high voltage.
    • Press the FIL button (on left next to the BEAM button) to activate the filament.
  • Slowly turn the condenser knob counterclockwise until the screen is lightly illuminated. Allow your eyes to adjust to the dark before making it any brighter.
  • Using the brightness knob and condenser knob, adjust the brightness of the image as needed, but not brighter than necessary (reason: a brighter beam means more electrons hitting the specimen which can be damaging). If holes begin to appear in the specimen and it appears to be melting, turn the condenser knob clockwise to dim the beam.
  • Use the trackball to move and view the sample.

Special thanks to Scott Taylor former FIT grad student for this great document and accompanying images.