3. While working at liquid nitrogen temperatures, clip specimen
grids into a cryo-FIB autogrid support. The grid should be
clipped so that the cell side (the carbon side) is facing towards
the flat surface of the autogrid so that cells are visible during
milling (see Note 3). Clipped grids may be kept for several
weeks in a liquid nitrogen storage dewar before subsequent
processing.
4. If fluorescent correlation is required for targeted FIB milling, it
should be collected beforehand.
When idle, our microscope is routinely kept in the following manner:
l
At room temperature, the microscope chamber is pumped
down to about 2 Â 10
À6 mbar.
l
Heat exchanger flow controller set to 1 L/min for both
stage and shield. This keeps the gas lines free of moisture.
l
Preferably, the sample transfer arm (Fig. 3c) is attached to
the microscope quickloader and pumped. This minimizes
dust and moisture contamination inside the transfer arm and
the quickloader.
l
Typically, we keep the FIB source heating is turned off if the
system is idle for more than a day, to conserve gallium.
Otherwise, the source may be left on for the next user.
However, practices may vary among facilities depending
on instrument usage frequency.
3.2.2 Day of Milling
Microscope Startup
and Cooldown
1. Turn on the FIB and SEM beams, typically done by “waking”
the microscope. Start the user interface and inspect the system
status as described in the following steps 2–4.
2. Check that the chamber base pressure is about 2 Â 10
À6 mbar
(see Note 4)
3. Check that the stage is empty.
4. Home the stage to reset stage coordinates and verify range of
motion.
5. Check gas nitrogen source pressure and adjust to 80 psi. If the
system is attached to a tank, check that the tank contains
sufficient nitrogen for the session. Replace if needed.
6. Begin purging the heat exchanger gas lines by opening the flow
controller to the maximum. Purge for at least 10 min.
7. Begin venting the transfer lid assembly (Fig. 3d). This step is
necessary to remove residual moisture inside the lines.
8. Prepare for loading: turn on the hot plate on the preparation
controller (Fig. 3h) and place required tools on it.
Practical Approaches for Cryo-FIB Milling
63
grids into a cryo-FIB autogrid support. The grid should be
clipped so that the cell side (the carbon side) is facing towards
the flat surface of the autogrid so that cells are visible during
milling (see Note 3). Clipped grids may be kept for several
weeks in a liquid nitrogen storage dewar before subsequent
processing.
4. If fluorescent correlation is required for targeted FIB milling, it
should be collected beforehand.
When idle, our microscope is routinely kept in the following manner:
l
At room temperature, the microscope chamber is pumped
down to about 2 Â 10
À6 mbar.
l
Heat exchanger flow controller set to 1 L/min for both
stage and shield. This keeps the gas lines free of moisture.
l
Preferably, the sample transfer arm (Fig. 3c) is attached to
the microscope quickloader and pumped. This minimizes
dust and moisture contamination inside the transfer arm and
the quickloader.
l
Typically, we keep the FIB source heating is turned off if the
system is idle for more than a day, to conserve gallium.
Otherwise, the source may be left on for the next user.
However, practices may vary among facilities depending
on instrument usage frequency.
3.2.2 Day of Milling
Microscope Startup
and Cooldown
1. Turn on the FIB and SEM beams, typically done by “waking”
the microscope. Start the user interface and inspect the system
status as described in the following steps 2–4.
2. Check that the chamber base pressure is about 2 Â 10
À6 mbar
(see Note 4)
3. Check that the stage is empty.
4. Home the stage to reset stage coordinates and verify range of
motion.
5. Check gas nitrogen source pressure and adjust to 80 psi. If the
system is attached to a tank, check that the tank contains
sufficient nitrogen for the session. Replace if needed.
6. Begin purging the heat exchanger gas lines by opening the flow
controller to the maximum. Purge for at least 10 min.
7. Begin venting the transfer lid assembly (Fig. 3d). This step is
necessary to remove residual moisture inside the lines.
8. Prepare for loading: turn on the hot plate on the preparation
controller (Fig. 3h) and place required tools on it.
Practical Approaches for Cryo-FIB Milling
63