3.2 Coma-Free
Alignment
In order to avoid the introduction of large phase errors at higher
resolution due to axial and off-axis coma, one must ensure that
illumination of the specimen occurs at an axis that is parallel to the
optical axis. This can be performed using the coma-free alignment
procedures described in Glaeser et al. 2011 [2] as implemented
within the Leginon [14] software (see Notes 22 and 23).
1. Move to an intact square on the calibration grid and ensure the
specimen is at eucentric height and proper focus.
2. Create a preset within Leginon [14] that uses the same magnification (e.g., SA 45,000Â in Nano probe mode) and beam
intensity settings as the exposure preset (e.g., C2 strength
43.5%). Set this preset to use a defocus of À300 nm and acquire
924 Â 924 (binned by 2) images with a two-second exposure
(see Note 24).
3. Go to the “Beam Tilt Image” node in Leginon [14] and set
these parameters in the settings tab.
l
Tableau Type: beam tilt series-power.
l
Beam Tilt: 0.005 radians.
l
Number of tilt directions: 4.
l
Start angle: 0.
l
Correlation type: phase.
l
Tableau binning: 2.
l
Beam tilt count: 1.
4. Click on the “Simulate target” icon in the Beam Tilt Image
node. Leginon [14] will now acquire a series of 5 images—a
zero-tilt image and 4 tilted images—with differing amounts of
beam tilt—5 milliradians of beam tilt in each “minus” X,
“plus” X, “minus” Y, and “plus” Y directions, respectively.
5. The FFTs of opposing images in the resulting tableau (i.e.,
“minus” Y and “plus” Y images) should have approximately
the same amount of astigmatism and Thon ring spacing. The
presence of slightly different astigmatism and/or number of
Thon rings in the FFTs of the “plus” versus “minus” tilt angles
demonstrates that the beam direction is not parallel to the
coma-free axis. To make adjustments, click on the zero-tilt
image in the direction towards the image(s) that is (are) further
from focus (i.e., FFT images with more Thon rings). If significant adjustments are necessary, it is recommended to repeat the
rotation center alignment (step 9, Subheading 3.1) and recenter the objective aperture (see Note 20). Repeat step 8 (Subheading 3.1) to ensure the objective lens is stigmated.
3.3 Parallel
Illumination
The parallel illumination condition for a two-condenser lens TEM
requires the source image to lie at the front focal plane of the upper
objective lens pole piece. As the objective lens is symmetrical,
134
Mark A. Herzik Jr
Alignment
In order to avoid the introduction of large phase errors at higher
resolution due to axial and off-axis coma, one must ensure that
illumination of the specimen occurs at an axis that is parallel to the
optical axis. This can be performed using the coma-free alignment
procedures described in Glaeser et al. 2011 [2] as implemented
within the Leginon [14] software (see Notes 22 and 23).
1. Move to an intact square on the calibration grid and ensure the
specimen is at eucentric height and proper focus.
2. Create a preset within Leginon [14] that uses the same magnification (e.g., SA 45,000Â in Nano probe mode) and beam
intensity settings as the exposure preset (e.g., C2 strength
43.5%). Set this preset to use a defocus of À300 nm and acquire
924 Â 924 (binned by 2) images with a two-second exposure
(see Note 24).
3. Go to the “Beam Tilt Image” node in Leginon [14] and set
these parameters in the settings tab.
l
Tableau Type: beam tilt series-power.
l
Beam Tilt: 0.005 radians.
l
Number of tilt directions: 4.
l
Start angle: 0.
l
Correlation type: phase.
l
Tableau binning: 2.
l
Beam tilt count: 1.
4. Click on the “Simulate target” icon in the Beam Tilt Image
node. Leginon [14] will now acquire a series of 5 images—a
zero-tilt image and 4 tilted images—with differing amounts of
beam tilt—5 milliradians of beam tilt in each “minus” X,
“plus” X, “minus” Y, and “plus” Y directions, respectively.
5. The FFTs of opposing images in the resulting tableau (i.e.,
“minus” Y and “plus” Y images) should have approximately
the same amount of astigmatism and Thon ring spacing. The
presence of slightly different astigmatism and/or number of
Thon rings in the FFTs of the “plus” versus “minus” tilt angles
demonstrates that the beam direction is not parallel to the
coma-free axis. To make adjustments, click on the zero-tilt
image in the direction towards the image(s) that is (are) further
from focus (i.e., FFT images with more Thon rings). If significant adjustments are necessary, it is recommended to repeat the
rotation center alignment (step 9, Subheading 3.1) and recenter the objective aperture (see Note 20). Repeat step 8 (Subheading 3.1) to ensure the objective lens is stigmated.
3.3 Parallel
Illumination
The parallel illumination condition for a two-condenser lens TEM
requires the source image to lie at the front focal plane of the upper
objective lens pole piece. As the objective lens is symmetrical,
134
Mark A. Herzik Jr