7. All tools used should be pre-cooled before contacting the
sample.
8. Place the autogrids into the shuttle with the cell side facing
up. Depending on which type of shuttle is used, you can load
1–2 autogrids at a time.
9. After attaching the transfer-unit’s insertion rod to the shuttle,
pump the vacuum in the slush chamber until liquid nitrogen
freezes. Lift out the shuttle into the transfer unit and vent the
slush chamber.
10. Attach the transfer unit to the airlock of the prep-chamber and
press “pump vacuum.” The vacuum has to reach a certain level
to safely open the airlock valves. A layer of platinum can be
sputter-coated in the prep-chamber to increase conductivity
and provide additional protection to the sample.
11. After the vacuum in the prep-chamber reaches the desired
level, open the valve between the prep-chamber and SEM.
Make sure the SEM cryo-stage is in the loading position.
Transfer the shuttle onto the cryo-stage with the insertion
rod and then retract the rod. The transfer unit should be
removed and the valves should be closed.
12. To begin milling lamellae, the electron and ion beams should
be in the operational state.
13. Move stage to image position and adjust focus and astigmatism
for the electron beam. Link Z to FWD after doing so.
14. Find a marker on the grid and adjust eucentric height so the ion
and electron beams point to the same location.
15. An additional layer of organometallic platinum can be deposited using the gas ingestion system (GIS) in the SEM. A
detailed description of the operation of the GIS can be found
in a protocol developed by the Baumeister lab [33].
16. The milling process usually starts with a higher current like
0.3 nA or 100 pA depending on the settings your FIB/SEM
has. Two rectangular areas several microns in width
(in mammalian cells, usually 10 μm) are drawn above and
below the target region. Leave 3–5 μm in between to avoid
damage by high-current ions.
17. Reduce the current to 30 pA and redraw the pattern of milling
to remove any material left. Leave ~1 μm between the two
patterns.
18. Use a current of 10 pA to do the final cleaning and reduce the
thickness of the lamella to 200–500 nm. For this last step of
milling, an additional 1
tilt can be added so the final thickness
of the lamella is more homogenous.
Methods in Cryo-Electron Tomography
91
sample.
8. Place the autogrids into the shuttle with the cell side facing
up. Depending on which type of shuttle is used, you can load
1–2 autogrids at a time.
9. After attaching the transfer-unit’s insertion rod to the shuttle,
pump the vacuum in the slush chamber until liquid nitrogen
freezes. Lift out the shuttle into the transfer unit and vent the
slush chamber.
10. Attach the transfer unit to the airlock of the prep-chamber and
press “pump vacuum.” The vacuum has to reach a certain level
to safely open the airlock valves. A layer of platinum can be
sputter-coated in the prep-chamber to increase conductivity
and provide additional protection to the sample.
11. After the vacuum in the prep-chamber reaches the desired
level, open the valve between the prep-chamber and SEM.
Make sure the SEM cryo-stage is in the loading position.
Transfer the shuttle onto the cryo-stage with the insertion
rod and then retract the rod. The transfer unit should be
removed and the valves should be closed.
12. To begin milling lamellae, the electron and ion beams should
be in the operational state.
13. Move stage to image position and adjust focus and astigmatism
for the electron beam. Link Z to FWD after doing so.
14. Find a marker on the grid and adjust eucentric height so the ion
and electron beams point to the same location.
15. An additional layer of organometallic platinum can be deposited using the gas ingestion system (GIS) in the SEM. A
detailed description of the operation of the GIS can be found
in a protocol developed by the Baumeister lab [33].
16. The milling process usually starts with a higher current like
0.3 nA or 100 pA depending on the settings your FIB/SEM
has. Two rectangular areas several microns in width
(in mammalian cells, usually 10 μm) are drawn above and
below the target region. Leave 3–5 μm in between to avoid
damage by high-current ions.
17. Reduce the current to 30 pA and redraw the pattern of milling
to remove any material left. Leave ~1 μm between the two
patterns.
18. Use a current of 10 pA to do the final cleaning and reduce the
thickness of the lamella to 200–500 nm. For this last step of
milling, an additional 1
tilt can be added so the final thickness
of the lamella is more homogenous.
Methods in Cryo-Electron Tomography
91