(c) Changes in magnification: Increasing the magnification
will decrease the measured exposure rate on the camera
but the flux should not change (see Note 30). Changes in
magnification only affect the projection system and
should not alter conditions for parallel illumination.
(d) Changes in gun lens: Increasing the gun lens (e.g., from
2 to 3) results in increased demagnification of the source
and to decreased current in the beam. In general, increasing the gun lens by 1 increment leads to a reduction in the
beam current by approximately 35%.
4. Using the determined exposure rate, adjust the total movie
recording time to yield a cumulative exposure of ~50–65 e
À /
A ˚ 2 (for single particle analysis) or ~80–100 e
À /A ˚ 2 (for tomography of thick specimens). Adjust the per-frame exposure time
(e.g., 100–250 ms) to yield a per-frame cumulative exposure of
~0.8 e
À /A ˚ 2 /frame or greater, while maximizing the number of
frames to limit the cumulative movement per frame to allow for
better correction of per-frame movement.
3.5 K2 Gain
Corrections
1. Set the microscope to a lower magnification (e.g., LM-280Â)
and use the trackball to move the stage to the center of an
empty area of the calibration grid.
2. Insert the K2 camera. In Digital Micrograph
® , go to Camera
and select “Prepare Gain Reference”. Follow prompts. Please
refer to the user guidelines for additional instructions.
3. It is recommended to collect new hardware dark references at
least once every 12–24 h of data collection to limit the deleterious effects of charge accumulation on the K2 sensor chip. The
frequency of hardware dark reference update increases with
lower acceleration voltages.
4 Notes
1. This protocol used: Ted Pella, Inc. Product # 677.
2. The steps outlined in this chapter are optimized for determining parallel illumination on the Talos Arctica; however, the
principle steps should apply to nearly all modern 2 condenser
lens electron microscopes with only minor modifications
necessary.
3. Alternative camera combinations will require slight modifications to the methods outlined in this chapter (e.g., magnifications used, camera interface) but the principle column
alignment steps should still apply.
4. It is recommended that only service engineers change the gun
settings on an X-FEG.
138
Mark A. Herzik Jr
will decrease the measured exposure rate on the camera
but the flux should not change (see Note 30). Changes in
magnification only affect the projection system and
should not alter conditions for parallel illumination.
(d) Changes in gun lens: Increasing the gun lens (e.g., from
2 to 3) results in increased demagnification of the source
and to decreased current in the beam. In general, increasing the gun lens by 1 increment leads to a reduction in the
beam current by approximately 35%.
4. Using the determined exposure rate, adjust the total movie
recording time to yield a cumulative exposure of ~50–65 e
À /
A ˚ 2 (for single particle analysis) or ~80–100 e
À /A ˚ 2 (for tomography of thick specimens). Adjust the per-frame exposure time
(e.g., 100–250 ms) to yield a per-frame cumulative exposure of
~0.8 e
À /A ˚ 2 /frame or greater, while maximizing the number of
frames to limit the cumulative movement per frame to allow for
better correction of per-frame movement.
3.5 K2 Gain
Corrections
1. Set the microscope to a lower magnification (e.g., LM-280Â)
and use the trackball to move the stage to the center of an
empty area of the calibration grid.
2. Insert the K2 camera. In Digital Micrograph
® , go to Camera
and select “Prepare Gain Reference”. Follow prompts. Please
refer to the user guidelines for additional instructions.
3. It is recommended to collect new hardware dark references at
least once every 12–24 h of data collection to limit the deleterious effects of charge accumulation on the K2 sensor chip. The
frequency of hardware dark reference update increases with
lower acceleration voltages.
4 Notes
1. This protocol used: Ted Pella, Inc. Product # 677.
2. The steps outlined in this chapter are optimized for determining parallel illumination on the Talos Arctica; however, the
principle steps should apply to nearly all modern 2 condenser
lens electron microscopes with only minor modifications
necessary.
3. Alternative camera combinations will require slight modifications to the methods outlined in this chapter (e.g., magnifications used, camera interface) but the principle column
alignment steps should still apply.
4. It is recommended that only service engineers change the gun
settings on an X-FEG.
138
Mark A. Herzik Jr