6. Insert the proper size of the C2 aperture. For electron diffraction on an FEI Tecnai TEM or a two-condenser lens system, a
small C2 aperture (50 μm) is recommended for a better beam
coherence.
7. Adjusting the C2 lens currents using the Intensity/Brightness
knob. Fine-tune the position of the C2 aperture until the beam
shrinks or expands its size to the screen center while adjusting
the Intensity/Brightness knob.
8. Align the beam shift/tilt of the condenser lens system.
9. Align the rotation center until the beam shrinks or expands its
size to the screen center.
10. Set a higher magnification to about 50,000Â. Go to step 8 and
repeat the alignment procedure a few cycles until the alignment
is stable.
11. Insert the objective aperture if one plans to perform electron
imaging on the 2D crystal samples. Switch to diffraction mode
and align the position of the objective aperture.
12. If the specimen is present, find an area with a flat carbon film
and adjust the eucentric height. Normalize the objective lenses
using the Eucentric Focus knob. Perform the coma-free
alignment.
3.2.3 Electron Diffraction
Electron diffraction is generally used when a well-ordered and large
2D crystal (in μm size range) is available. The intensity data can be
accurately recorded without CTF modulation.
1. Align the beam condition to obtain a parallel illumination. In a
two-condenser lens system, a small condenser aperture (50 μm)
and the smallest spot size (spot size step 11) are recommended
to use.
2. In the Low Dose system, go to the Exposure mode. Find the
right focus and centered the beam. Make sure that the objective
aperture is out of the path.
3. Set the magnification to about 50,000Â and spread out the
beam to obtain a target dose rate of about 0.3 e
À /A ˚ 2 /s.
4. Switch to the diffraction mode. Set an appropriate camera
length for diffraction experiments (see Note 12).
5. Align the beam to the screen center using a diffraction lens
shift. Adjust the beam shape to a circle with the diffraction lens
astigmatism.
6. Focus the beam using the Diffraction/Focus knob.
7. Go to Search mode and set the magnification to the smallest in
the SA mode. Set the Search mode in the diffraction mode. Use
the same camera length as that in the Exposure mode. Overfocus to spread the beam until the crystal can be recognized.
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small C2 aperture (50 μm) is recommended for a better beam
coherence.
7. Adjusting the C2 lens currents using the Intensity/Brightness
knob. Fine-tune the position of the C2 aperture until the beam
shrinks or expands its size to the screen center while adjusting
the Intensity/Brightness knob.
8. Align the beam shift/tilt of the condenser lens system.
9. Align the rotation center until the beam shrinks or expands its
size to the screen center.
10. Set a higher magnification to about 50,000Â. Go to step 8 and
repeat the alignment procedure a few cycles until the alignment
is stable.
11. Insert the objective aperture if one plans to perform electron
imaging on the 2D crystal samples. Switch to diffraction mode
and align the position of the objective aperture.
12. If the specimen is present, find an area with a flat carbon film
and adjust the eucentric height. Normalize the objective lenses
using the Eucentric Focus knob. Perform the coma-free
alignment.
3.2.3 Electron Diffraction
Electron diffraction is generally used when a well-ordered and large
2D crystal (in μm size range) is available. The intensity data can be
accurately recorded without CTF modulation.
1. Align the beam condition to obtain a parallel illumination. In a
two-condenser lens system, a small condenser aperture (50 μm)
and the smallest spot size (spot size step 11) are recommended
to use.
2. In the Low Dose system, go to the Exposure mode. Find the
right focus and centered the beam. Make sure that the objective
aperture is out of the path.
3. Set the magnification to about 50,000Â and spread out the
beam to obtain a target dose rate of about 0.3 e
À /A ˚ 2 /s.
4. Switch to the diffraction mode. Set an appropriate camera
length for diffraction experiments (see Note 12).
5. Align the beam to the screen center using a diffraction lens
shift. Adjust the beam shape to a circle with the diffraction lens
astigmatism.
6. Focus the beam using the Diffraction/Focus knob.
7. Go to Search mode and set the magnification to the smallest in
the SA mode. Set the Search mode in the diffraction mode. Use
the same camera length as that in the Exposure mode. Overfocus to spread the beam until the crystal can be recognized.
Electron Crystallography of Membrane Proteins
259