2. Switch to “high-resolution” mode in the fluorescent screen
viewer. If needed, adjust the brightness of the fluorescent
screen camera until the edge of the objective aperture is clearly
visible (Fig. 5b). Note that both the objective aperture and
gold powder diffraction rings are blurry. Bring the back focal
plane of the objective lens into view (i.e., to the object plane of
the diffraction lens) by adjusting diffraction focus using the
Focus knob until the edge of the objective aperture is as sharp/
crisp as possible (Fig. 5c) (see Note 25). If the gold powder
diffraction rings are not crisp, then the source image is not
located in the front focal plane of the objective lens and the
specimen is not illuminated using a parallel beam.
3. Adjust the beam intensity (i.e., C2 strength) until the width of
the gold powder diffraction rings is minimized (Fig. 5d).
4. If the objective aperture is no longer crisp, repeat steps 2 and
3 (Subheading 3.2) until both the edge of objective aperture is
crisp, and the width of the gold powder diffraction rings are
minimized (see Note 27).
5. Center the objective aperture (see step 7, Subheading 3.1).
6. Increase the diffraction camera length to D 5.7 m or until just
the central spot is visible (see Note 26). If needed, adjust the
brightness of the fluorescent camera screen until the shape of
the central spot is clearly visible (see Fig. 5e).
7. In the Tecnai User Interface, click on the “Diffraction” tab in
the Stigmator panel and use the multifunction X and Y knobs
to adjust the shape of the central spot while cycling above and
below the crossover point to make the beam circular. Iterate
until the beam is properly stigmated (i.e., the beam shape is
similar on both sides of crossover) (see Fig. 5f).
8. Adjust the beam intensity until the central spot is minimized
(Fig. 5g). This will align the source image to the front focal
plane of the upper objective lens, establishing the parallel illumination condition. Make note of the beam intensity value,
as this is the only value under the current microscope settings that confers parallel illumination, and should be used
for exposure image acquisition (see Note 28). Important:
adjustments to the exposure rate must be performed
through changes in spot size, C2 aperture, gun lens, or
magnification rather than changes in beam intensity.
9. Decrease the camera length to D 850 mm and ensure that the
central spot is centered, and the objective aperture is centered
(Fig. 5h).
10. Exit Diffraction Mode and center the beam on the camera by
performing beam shift alignment in the Tecnai User Interface
Direct Alignments. Ensure that the objective lens is properly
stigmated (see step 7, Subheading 3.1).
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Mark A. Herzik Jr
viewer. If needed, adjust the brightness of the fluorescent
screen camera until the edge of the objective aperture is clearly
visible (Fig. 5b). Note that both the objective aperture and
gold powder diffraction rings are blurry. Bring the back focal
plane of the objective lens into view (i.e., to the object plane of
the diffraction lens) by adjusting diffraction focus using the
Focus knob until the edge of the objective aperture is as sharp/
crisp as possible (Fig. 5c) (see Note 25). If the gold powder
diffraction rings are not crisp, then the source image is not
located in the front focal plane of the objective lens and the
specimen is not illuminated using a parallel beam.
3. Adjust the beam intensity (i.e., C2 strength) until the width of
the gold powder diffraction rings is minimized (Fig. 5d).
4. If the objective aperture is no longer crisp, repeat steps 2 and
3 (Subheading 3.2) until both the edge of objective aperture is
crisp, and the width of the gold powder diffraction rings are
minimized (see Note 27).
5. Center the objective aperture (see step 7, Subheading 3.1).
6. Increase the diffraction camera length to D 5.7 m or until just
the central spot is visible (see Note 26). If needed, adjust the
brightness of the fluorescent camera screen until the shape of
the central spot is clearly visible (see Fig. 5e).
7. In the Tecnai User Interface, click on the “Diffraction” tab in
the Stigmator panel and use the multifunction X and Y knobs
to adjust the shape of the central spot while cycling above and
below the crossover point to make the beam circular. Iterate
until the beam is properly stigmated (i.e., the beam shape is
similar on both sides of crossover) (see Fig. 5f).
8. Adjust the beam intensity until the central spot is minimized
(Fig. 5g). This will align the source image to the front focal
plane of the upper objective lens, establishing the parallel illumination condition. Make note of the beam intensity value,
as this is the only value under the current microscope settings that confers parallel illumination, and should be used
for exposure image acquisition (see Note 28). Important:
adjustments to the exposure rate must be performed
through changes in spot size, C2 aperture, gun lens, or
magnification rather than changes in beam intensity.
9. Decrease the camera length to D 850 mm and ensure that the
central spot is centered, and the objective aperture is centered
(Fig. 5h).
10. Exit Diffraction Mode and center the beam on the camera by
performing beam shift alignment in the Tecnai User Interface
Direct Alignments. Ensure that the objective lens is properly
stigmated (see step 7, Subheading 3.1).
136
Mark A. Herzik Jr