is only unlocked for about 90 s before the action times out and
relocks. The valve must be opened within this timeframe. When
closing the valve, wait for it to lock before reopening or
re-issuing the pump command. Otherwise, we have found
that this may lead to the gate valve being stuck in the open
state.
7. Setting the scan rotation to 180
is optional, but it orients the
SEM and FIB views to appear more natural such that the FIB
view shows cells “sitting” on the grid, rather than “hanging”
from the grid. In some cases, the scan rotation of the FIB
column is not perfectly parallel to the grid surface. In this
case, the resulting lamellae will be tilted relative to the milling
axis (Fig. 9a–d). Such tilted lamellae become more difficult for
tomography due to limited focus and tracking area (see Subheading 3.3). This issue can be corrected by rotating the scan
direction of the FIB column until the grid surface appears
horizontal (Fig. 9e–h).
8. In addition to sample specific considerations for milling, users
should consider the accessible area of the grid at the TEM for
tilt series acquisition. This depends on the specific TEM instrument, but is typically limited to the grid squares that are in the
center 1 mm of the grid. For a 200 mesh grid, this is about
10-by-10 grid squares centered over the grid. Lamellae should
be milled within this region. To minimize occlusion from the
grid bars at high tilt at the TEM, it is generally best to make
lamellae that are centered in the grid square.
9. Sputter coating is used to improve the image quality of
non-conductive samples by depositing a layer of metal to
allow charge to quickly dissipate. The Aquilos has a built-in
sputter coater. Set the number of purge cycles to 0 (which
would otherwise lead to sample contamination) and click “Prepare for Sputtering.” The microscope will enter a low vacuum
state (about 0.1 mbar) by partially venting with dry argon, and
the stage temperature will increase slightly to about À170
C.
The stage will move to the sputtering position. When the
preparation actions have finished, select the sputtering parameters or the preset values and run the process. Typical sputter
conditions for initial rough sputtering are 1 kV, 30 mA, for 15 s
at 0.10 mbar chamber pressure. You can see the plasma glow
using the chamber camera. When sputtering is complete, click
“Recover from Sputtering” to return the stage to the mapping
position and return to high vacuum
10. The GIS deposition time depends on the sample type, the
intended deposition thickness, and the preset deposition
stage coordinate. If the stage is closer to the tip of the GIS
needle, a shorter deposition time will yield the same thickness
78
Vinson Lam and Elizabeth Villa
relocks. The valve must be opened within this timeframe. When
closing the valve, wait for it to lock before reopening or
re-issuing the pump command. Otherwise, we have found
that this may lead to the gate valve being stuck in the open
state.
7. Setting the scan rotation to 180
is optional, but it orients the
SEM and FIB views to appear more natural such that the FIB
view shows cells “sitting” on the grid, rather than “hanging”
from the grid. In some cases, the scan rotation of the FIB
column is not perfectly parallel to the grid surface. In this
case, the resulting lamellae will be tilted relative to the milling
axis (Fig. 9a–d). Such tilted lamellae become more difficult for
tomography due to limited focus and tracking area (see Subheading 3.3). This issue can be corrected by rotating the scan
direction of the FIB column until the grid surface appears
horizontal (Fig. 9e–h).
8. In addition to sample specific considerations for milling, users
should consider the accessible area of the grid at the TEM for
tilt series acquisition. This depends on the specific TEM instrument, but is typically limited to the grid squares that are in the
center 1 mm of the grid. For a 200 mesh grid, this is about
10-by-10 grid squares centered over the grid. Lamellae should
be milled within this region. To minimize occlusion from the
grid bars at high tilt at the TEM, it is generally best to make
lamellae that are centered in the grid square.
9. Sputter coating is used to improve the image quality of
non-conductive samples by depositing a layer of metal to
allow charge to quickly dissipate. The Aquilos has a built-in
sputter coater. Set the number of purge cycles to 0 (which
would otherwise lead to sample contamination) and click “Prepare for Sputtering.” The microscope will enter a low vacuum
state (about 0.1 mbar) by partially venting with dry argon, and
the stage temperature will increase slightly to about À170
C.
The stage will move to the sputtering position. When the
preparation actions have finished, select the sputtering parameters or the preset values and run the process. Typical sputter
conditions for initial rough sputtering are 1 kV, 30 mA, for 15 s
at 0.10 mbar chamber pressure. You can see the plasma glow
using the chamber camera. When sputtering is complete, click
“Recover from Sputtering” to return the stage to the mapping
position and return to high vacuum
10. The GIS deposition time depends on the sample type, the
intended deposition thickness, and the preset deposition
stage coordinate. If the stage is closer to the tip of the GIS
needle, a shorter deposition time will yield the same thickness
78
Vinson Lam and Elizabeth Villa