9. Pump the transfer lid airlock for 35–40 s (make sure transfer lid
sliding valve is closed), then open the transfer arm valve. The
next three steps should be done quickly in succession to
minimize contamination.
10. Open the transfer arm valve, then vent the transfer lid airlock
for 3 s. Stop venting.
11. Unlock the transfer arm.
12. Open the transfer lid slider and quickly and gently insert the
transfer arm to dock the shuttle into the loading station.
13. Release the shuttle grabber and retract the arm. If desired,
pump down the transfer arm as described above.
14. Remove the transfer lid from the loading station and replace
with the standard lid.
15. Using pre-cooled tools, flip the shuttle into the horizontal
loading position, turn the locking screw to release the grids,
then transfer the grids to a pre-cooled, labeled grid box.
16. Store the grid boxes with your specimens in liquid nitrogen.
End of Session Tasks
1. Remove heat exchanger from dewar and allow the stage and
shield to warm to room temperature (30–60 min)
2. Home the stage for the next user.
3. If the microscope will not be used in the next day, sleep the
system to conserve gallium. Otherwise, keep it on but ensure
the SEM and FIB column valves are closed.
4. Turn off hot plates and store tools.
5. When the stage and shield are near room temperature, turn the
flow rates to 1 L/min to prevent moisture accumulation inside
the heat exchanger gas lines.
3.3 Considerations
for TEM
The specimen should be loaded such that the FIB milling axis is
perpendicular to the TEM tilt axis (Fig. 7). This will allow maximum tilt range for tomography without the unmilled material
blocking the field of view at high tilts. Due to FIB milling geometry, the lamella will have a measurable pre-tilt at the TEM ranging
from 5
to 15
difference compared to reported stage goniometer
angle. This pre-tilt will influence the tilt series acquisition scheme
because in one tilt direction the specimen will appear to be much
thicker at high tilt angles compared to the other direction (see
Subheading 1.2). Additionally, sample orientation is important
for accurate focus and tracking when acquiring data with
low-dose techniques. TEM data acquisition is mostly handled
through automated routines from software packages such as SerialEM [44, 45].
Practical Approaches for Cryo-FIB Milling
71
sliding valve is closed), then open the transfer arm valve. The
next three steps should be done quickly in succession to
minimize contamination.
10. Open the transfer arm valve, then vent the transfer lid airlock
for 3 s. Stop venting.
11. Unlock the transfer arm.
12. Open the transfer lid slider and quickly and gently insert the
transfer arm to dock the shuttle into the loading station.
13. Release the shuttle grabber and retract the arm. If desired,
pump down the transfer arm as described above.
14. Remove the transfer lid from the loading station and replace
with the standard lid.
15. Using pre-cooled tools, flip the shuttle into the horizontal
loading position, turn the locking screw to release the grids,
then transfer the grids to a pre-cooled, labeled grid box.
16. Store the grid boxes with your specimens in liquid nitrogen.
End of Session Tasks
1. Remove heat exchanger from dewar and allow the stage and
shield to warm to room temperature (30–60 min)
2. Home the stage for the next user.
3. If the microscope will not be used in the next day, sleep the
system to conserve gallium. Otherwise, keep it on but ensure
the SEM and FIB column valves are closed.
4. Turn off hot plates and store tools.
5. When the stage and shield are near room temperature, turn the
flow rates to 1 L/min to prevent moisture accumulation inside
the heat exchanger gas lines.
3.3 Considerations
for TEM
The specimen should be loaded such that the FIB milling axis is
perpendicular to the TEM tilt axis (Fig. 7). This will allow maximum tilt range for tomography without the unmilled material
blocking the field of view at high tilts. Due to FIB milling geometry, the lamella will have a measurable pre-tilt at the TEM ranging
from 5
to 15
difference compared to reported stage goniometer
angle. This pre-tilt will influence the tilt series acquisition scheme
because in one tilt direction the specimen will appear to be much
thicker at high tilt angles compared to the other direction (see
Subheading 1.2). Additionally, sample orientation is important
for accurate focus and tracking when acquiring data with
low-dose techniques. TEM data acquisition is mostly handled
through automated routines from software packages such as SerialEM [44, 45].
Practical Approaches for Cryo-FIB Milling
71