11. Measure the diameter of the beam using the measurement tool
in the Tecnai User Interface or by using the calibration grid
waffles as a reference. If the beam diameter is too large for the
desired data collection strategy, then switch to a smaller C2
aperture (see Note 29). If the smallest C2 aperture is already
selected, please adjust the data collection strategy accordingly
to ensure areas of the grid will not be double-exposed during
data collection. This is particularly important when using
Micro probe mode or larger diameter C2 apertures (Fig. 4)
(see Note 10). Experience has shown that the approximate
diameter of a parallel beam at SA 45,000Â (Nano probe
mode, 70 μm C2 aperture, gun lens 8, spot size 3, 2000 μm
C1 aperture, 100 μm objective aperture) is approximately
1.8 μm.
3.4 Determine
and Set
the Exposure Rate
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 (i.e., a large crack or empty
square).
2. Insert the K2 camera. In Digital Micrograph
® , click on the
settings icon in the “Camera View” window, making sure that
the binning is set to 1 and that the entire sensor is being used
by clicking on “Full CCD”. Set an exposure time of 1 second
and click on “start view”. The exposure rate (in e
À /pixel/s)
will be displayed in the Camera Monitor window. A measured
exposure rate of 2–5 e
À
/pixel/s is optimal for the K2 (see
Note 31).
3. If the measured exposure rate is not ideal for the current
experiment and adjustments to the exposure rate are required,
adjustments must be performed by changing spot size, C2
aperture, magnification, and/or guns lens rather than changing beam intensity. If changes in spot size, C2 aperture, and/or
gun lens are made, then the above-outlined steps should be
checked to ensure the scope has not deviated from the parallel
condition.
(a) Changes to spot size: Lowering the spot number (i.e.,
from 3 to 2) will, under most conditions, result in an
approximate doubling of the measured exposure rate.
On the contrary, increasing the spot number (i.e., from
spot size 3 to 4) will result in an approximate two-fold
decrease in measured exposure rate (see Notes 28 and 32).
(b) Changing to C2 aperture: Using a larger C2 aperture will
allow more electrons to illuminate the specimen and will
therefore increase the exposure rate. Likewise, using a
smaller C2 aperture will decrease the number of electrons
illuminating the specimen and will therefore decrease the
exposure rate (see Note 29).
Parallel Illumination on the Talos Arctica
137
in the Tecnai User Interface or by using the calibration grid
waffles as a reference. If the beam diameter is too large for the
desired data collection strategy, then switch to a smaller C2
aperture (see Note 29). If the smallest C2 aperture is already
selected, please adjust the data collection strategy accordingly
to ensure areas of the grid will not be double-exposed during
data collection. This is particularly important when using
Micro probe mode or larger diameter C2 apertures (Fig. 4)
(see Note 10). Experience has shown that the approximate
diameter of a parallel beam at SA 45,000Â (Nano probe
mode, 70 μm C2 aperture, gun lens 8, spot size 3, 2000 μm
C1 aperture, 100 μm objective aperture) is approximately
1.8 μm.
3.4 Determine
and Set
the Exposure Rate
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 (i.e., a large crack or empty
square).
2. Insert the K2 camera. In Digital Micrograph
® , click on the
settings icon in the “Camera View” window, making sure that
the binning is set to 1 and that the entire sensor is being used
by clicking on “Full CCD”. Set an exposure time of 1 second
and click on “start view”. The exposure rate (in e
À /pixel/s)
will be displayed in the Camera Monitor window. A measured
exposure rate of 2–5 e
À
/pixel/s is optimal for the K2 (see
Note 31).
3. If the measured exposure rate is not ideal for the current
experiment and adjustments to the exposure rate are required,
adjustments must be performed by changing spot size, C2
aperture, magnification, and/or guns lens rather than changing beam intensity. If changes in spot size, C2 aperture, and/or
gun lens are made, then the above-outlined steps should be
checked to ensure the scope has not deviated from the parallel
condition.
(a) Changes to spot size: Lowering the spot number (i.e.,
from 3 to 2) will, under most conditions, result in an
approximate doubling of the measured exposure rate.
On the contrary, increasing the spot number (i.e., from
spot size 3 to 4) will result in an approximate two-fold
decrease in measured exposure rate (see Notes 28 and 32).
(b) Changing to C2 aperture: Using a larger C2 aperture will
allow more electrons to illuminate the specimen and will
therefore increase the exposure rate. Likewise, using a
smaller C2 aperture will decrease the number of electrons
illuminating the specimen and will therefore decrease the
exposure rate (see Note 29).
Parallel Illumination on the Talos Arctica
137