imperfections (e.g., large cracks, broken areas, foil separated
from the grid bar). This area will be used for all column alignments outlined below.
2. Switch to a higher magnification (e.g., SA 11,000Â) in Nano
probe mode and bring the specimen to eucentric height using
any of the following methods:
(a) Choose a recognizable image feature (e.g., large gold
particle or obvious grid feature) and place that feature at
the center of the fluorescent screen. Switch on the Alpha
wobbler (Æ15
tilts) and minimize image movement by
adjusting specimen height (Z-shift). Iterate until the
image no longer moves (see Note 11).
(b) Tilt the stage to a defined angle (e.g., À25
) and adjust
the Z-height of the specimen until that feature is recentered on the fluorescent screen. Reset the stage back to
0
tilt and recenter the feature on the fluorescent screen.
Iterate until the image no longer moves. Larger stage tilts
can also be used to fine-tune the accuracy of eucentric
height (see Note 12).
(c) Press the Eucentric Focus button on the hand panel to
bring the focus level to, or very close to, eucentric height.
Focus the beam to a spot and adjust the height of the
specimen (Z-height) until the caustic ring is minimized to
a spot (see Notes 13 and 14).
(d) Use the auto-eucentric feature built into an automated
data collection software package (e.g., Leginon [14],
EPU [16], SerialEM [15]).
3. Press the Eucentric Focus button on the hand panel. Switch to
a higher magnification (e.g., SA 45,000Â) in Nano probe
mode and use the Intensity knob on the hand panel to spread
the beam (right side of crossover or clockwise) until the diameter of the beam is slightly larger than the 40 mm circle on the
fluorescent screen. With the objective aperture removed, use
Digital Micrograph® to display the Fourier Transform (FFT)
of a live image and determine proper focus of the specimen by
adjusting the Focus knob until the observed Thon rings in the
FFT are eliminated. Proper focus can also be determined using
automated data collection software packages (e.g., Leginon
[14], EPU [16], SerialEM [15]), but it is always best practice
to manually confirm proper focus has been determined by
viewing the live FFT in Digital Micrograph® (see Notes 15
and 16). After determining proper focus, press the “Reset
Defocus” button on the hand panel.
4. Select and center the 70 μm C2 aperture by first focusing the
beam to a spot and then using beam shift to center the focused
beam on the fluorescent screen. Expand the beam to the
40 mm circle. Click on the “Adjust” tab for the C2 aperture
132
Mark A. Herzik Jr
from the grid bar). This area will be used for all column alignments outlined below.
2. Switch to a higher magnification (e.g., SA 11,000Â) in Nano
probe mode and bring the specimen to eucentric height using
any of the following methods:
(a) Choose a recognizable image feature (e.g., large gold
particle or obvious grid feature) and place that feature at
the center of the fluorescent screen. Switch on the Alpha
wobbler (Æ15
tilts) and minimize image movement by
adjusting specimen height (Z-shift). Iterate until the
image no longer moves (see Note 11).
(b) Tilt the stage to a defined angle (e.g., À25
) and adjust
the Z-height of the specimen until that feature is recentered on the fluorescent screen. Reset the stage back to
0
tilt and recenter the feature on the fluorescent screen.
Iterate until the image no longer moves. Larger stage tilts
can also be used to fine-tune the accuracy of eucentric
height (see Note 12).
(c) Press the Eucentric Focus button on the hand panel to
bring the focus level to, or very close to, eucentric height.
Focus the beam to a spot and adjust the height of the
specimen (Z-height) until the caustic ring is minimized to
a spot (see Notes 13 and 14).
(d) Use the auto-eucentric feature built into an automated
data collection software package (e.g., Leginon [14],
EPU [16], SerialEM [15]).
3. Press the Eucentric Focus button on the hand panel. Switch to
a higher magnification (e.g., SA 45,000Â) in Nano probe
mode and use the Intensity knob on the hand panel to spread
the beam (right side of crossover or clockwise) until the diameter of the beam is slightly larger than the 40 mm circle on the
fluorescent screen. With the objective aperture removed, use
Digital Micrograph® to display the Fourier Transform (FFT)
of a live image and determine proper focus of the specimen by
adjusting the Focus knob until the observed Thon rings in the
FFT are eliminated. Proper focus can also be determined using
automated data collection software packages (e.g., Leginon
[14], EPU [16], SerialEM [15]), but it is always best practice
to manually confirm proper focus has been determined by
viewing the live FFT in Digital Micrograph® (see Notes 15
and 16). After determining proper focus, press the “Reset
Defocus” button on the hand panel.
4. Select and center the 70 μm C2 aperture by first focusing the
beam to a spot and then using beam shift to center the focused
beam on the fluorescent screen. Expand the beam to the
40 mm circle. Click on the “Adjust” tab for the C2 aperture
132
Mark A. Herzik Jr