moved, the electron path through the objective lens has
changed and therefore the objective aperture will no longer
be properly centered. To ensure that ideal imaging conditions
are maintained, and high-spatial frequencies are not inadvertently eliminated, verify the objective aperture is centered after
making changes to the rotation center alignment.
21. A misalignment of rotation center will result in an apparent
translation of the image upon increasing defocus.
22. Coma-free alignment can also be performed using Tecnai User
Interface, SerialEM [15], and/or Auto-CTF within EPU
[16]. Please refer to the respective user manual for detailed
instructions.
23. Coma-free alignment effectively “fine-tunes” the rotation center alignment performed in step 9 (Subheading 3.1). If this
alignment was performed correctly, then only minor adjustments will be required. Please refer to Note 20 when making
adjustments to ensure coma-free alignment.
24. It is recommended to adjust the defocus (e.g., 300–500 nm
underfocus) to observe only 1–2 Thon rings in the tableau FFT
images. Large underfocus (i.e., >1000 nm) will result in too
many Thon rings and will make it more difficult to assess
astigmatism and differences between the negatively and positively tilted images.
25. Making adjustments to focus in diffraction mode does not
physically move the objective aperture but rather places the
image plane in the back focal plane of the objective lens (i.e.,
where the objective aperture is physically located).
26. The exact camera length in Diffraction Mode will vary for each
microscope depending on camera and imaging accessory
combinations.
27. If only a portion of the objective aperture is crisp (i.e., crisp left
and right edges versus blurry top and bottom edges), or the
width of the gold powder diffraction rings varies around the
central spot, then the diffraction lens is not properly stigmated.
Follow steps 6–9 (Subheading 3.3).
28. It is good practice to keep track of C2 intensity values for
parallel illumination under varying combinations of spot size,
C2 aperture, and gun lens, as significant deviations between
data collections indicate a potential misalignment of the
column.
29. If the C2 aperture is properly centered and stigmated, and the
spot size remains unchanged, then the C2 intensity value for
parallel illumination should remain unchanged as well. However, it is still recommended to verify the parallel illumination
condition is preserved when changing between C2 apertures
(refer to Subheading 3.3).
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Mark A. Herzik Jr
changed and therefore the objective aperture will no longer
be properly centered. To ensure that ideal imaging conditions
are maintained, and high-spatial frequencies are not inadvertently eliminated, verify the objective aperture is centered after
making changes to the rotation center alignment.
21. A misalignment of rotation center will result in an apparent
translation of the image upon increasing defocus.
22. Coma-free alignment can also be performed using Tecnai User
Interface, SerialEM [15], and/or Auto-CTF within EPU
[16]. Please refer to the respective user manual for detailed
instructions.
23. Coma-free alignment effectively “fine-tunes” the rotation center alignment performed in step 9 (Subheading 3.1). If this
alignment was performed correctly, then only minor adjustments will be required. Please refer to Note 20 when making
adjustments to ensure coma-free alignment.
24. It is recommended to adjust the defocus (e.g., 300–500 nm
underfocus) to observe only 1–2 Thon rings in the tableau FFT
images. Large underfocus (i.e., >1000 nm) will result in too
many Thon rings and will make it more difficult to assess
astigmatism and differences between the negatively and positively tilted images.
25. Making adjustments to focus in diffraction mode does not
physically move the objective aperture but rather places the
image plane in the back focal plane of the objective lens (i.e.,
where the objective aperture is physically located).
26. The exact camera length in Diffraction Mode will vary for each
microscope depending on camera and imaging accessory
combinations.
27. If only a portion of the objective aperture is crisp (i.e., crisp left
and right edges versus blurry top and bottom edges), or the
width of the gold powder diffraction rings varies around the
central spot, then the diffraction lens is not properly stigmated.
Follow steps 6–9 (Subheading 3.3).
28. It is good practice to keep track of C2 intensity values for
parallel illumination under varying combinations of spot size,
C2 aperture, and gun lens, as significant deviations between
data collections indicate a potential misalignment of the
column.
29. If the C2 aperture is properly centered and stigmated, and the
spot size remains unchanged, then the C2 intensity value for
parallel illumination should remain unchanged as well. However, it is still recommended to verify the parallel illumination
condition is preserved when changing between C2 apertures
(refer to Subheading 3.3).
142
Mark A. Herzik Jr