That being said, I've decided to write up a procedure for alignment on the Tecnai in as much detail as I could. I am not an experienced user by any stretch of the word, and I do suggest that you use what I've provided here in conjunction with your own notes from your training session, but I hope that these steps can help you in some way.
Please note that this procedure only applies to basic imaging on the Tecnai (I know, I know, these steps look anything but "basic.") Other techniques, such as electron diffraction or electron energy-loss spectroscopy, may require additional steps. I'll be sure to update this post when I eventually learn how to do those!
Tecnai – Standard Operating Procedure
1. Make sure that the condenser aperture is in and the objective aperture and selected area aperture are out. Check that the column valve is closed, high tension is on, and the filament is off. Gun should be < 25 and column pressure should be < 15. Note: Yellow button indicates that it is “active” and a grey button indicates that it is not active. For the objective aperture, 6 or 7 means "out." For the selected area aperture, pin to the right means “out” and to the left means “in.”
2. Fill up the liquid nitrogen dewar if it is not already filled.
3. Reset the holder by going to Search > Stage |>| Reset Holder
4. Load your sample
a. To take the sample holder out, place two fingers (left hand) on the purple ring. With your right hand, pull straight out and then turn clockwise until it stops. Then pull entire sample holder out gently.
b. Put sample into the holder with film side down.
c. To put the sample holder back in, put the rod in with pin at 3:00 until it hits the back plate. Then turn clockwise until the pin slides into the 4:00 slot. Wait for it to pump down for 1 minute.
5. Select single-tilt holder (the red light will go off).
6. Now you can turn the filament on. The target emission should be 6 – 10 microamps and recommended spot size is 3. It will take about a minute for the filament to heat up.
7. Open the column valve.
8. Press the eucentric focus button.
9. At about 34000x, find the eucentric height. This can be done by changing the z height until the point of minimum contrast is found, or by using the alpha wobbler to find the height at which the particles barely move as the stage tilts. Another way to find the eucentric height is by going to low magnification, condensing the beam (you should see diffraction rings now), and changing the z height until the rings collapse.
10. Align the condenser aperture (use aperture 3). Use the x and y knobs on the aperture (the side knob and the middle front knob) until the beam doesn’t swing when you change the intensity.
11. At around 78000x, check that the beam is round. If it is not, adjust the condenser stigmator by going to the Tune tab and selecting “condenser,” pressing the stigmator button, and using the multifunction knobs.
12. Condense the beam. In the Tune tab, check beam tilt x, beam tilt y, beam shift, and rotation center.
a. Beam tilt x and y: use multifunction knobs to make one main beam. Press “done” when finished.
b. Beam shift: use multifunction knobs to move beam to center. Press “done.”
c. Rotation center: Minimize the motion of particles (or make sure the beam flashes in and out only and not side to side). Press “done.”
13. If the objective aperture is needed, go into diffraction mode and use the multifunction knobs to center the beam. Select and insert the desired objective aperture, then center the diffraction pattern around the central spot using the aperture x and y knobs. Return to imaging mode.
14. At around 310kx, use the intensity knob to spread the beam to fill the screen.
15. Lift the screen.
16. Under the Camera tab, select BM Ultrascan > Insert. Then turn Search on.
17. In Digital Micrograph, go to Process > Live > FFT
18. Adjust the focus knob until you can see rings in the FFT. (Note: if the focus knob doesn't seem to change much, make sure the focus step size is big enough. This can be adjusted using the outer ring of the focus knob.) Adjust the objective stigmation (Choose “objective” in the Tune tab, press the stigmator button, and use the multifunction knobs) until the rings are round.
19. Press the “acquire” button to take an image. The folder that these images are saved to can be determined by going to File > Global info > save numbered > choose your folder.
20. At the end of your session, make sure that the objective and selected area apertures are out. Also make sure that the camera is out.
21. Make sure you are in imaging mode and that the screen is down.
22. Reset the holder by going to Search |>| holder > reset holder
23. Close the column valve.
24. Turn the filament off.
25. Take the sample holder out, remove your sample, and put the sample holder back in.
26. If there is someone on the Tecnai after you, top off the liquid nitrogen for them.
27. If not, take the dewar out and go to Vacuum |>| Cryo cycle
- Filling up the liquid nitrogen dewar in the beginning creates a cold trap which prevents gases and particles from contaminating your sample.
- Making sure that the column valve is closed before you load your sample ensures that the electron gun is protected in case the column vacuum is dumped.
- Pressing the eucentric focus button presets the objective lens to be focused at the eucentric height.
- Finding the eucentric height consequently brings your sample to that plane.
- When adjusting the condenser aperture so that the beam no longer swings as you change the intensity, you are centering the condenser aperture.
- Aligning the condenser stigmator corrects for astigmatism in the condenser lens.
- The beam tilt x, beam tilt y, beam shift, and rotation center checks all contribute to aligning the objective lens and differentiating tilts from shifts.
- Centering the diffraction pattern after inserting the objective aperture centers the objective aperture.
- And finally, making sure the rings are round in the FFT corrects for astigmatism in the objective lenses.
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