9. Fill a 4-L dewar with clean liquid nitrogen for sample preparation. Make sure the nitrogen hose attached to the source is dry
before filling to minimize ice contamination. Be sure to use
appropriate PPE when handling cryogenic liquids.
10. Fill the heat exchanger tank with liquid nitrogen.
11. Start the temperature logging software to monitor and record
stage and shield temperature.
12. Slowly insert the heat exchanger into the heat exchanger tank
and ensure that it is seated appropriately. Monitor the stage and
shield temperature until it stabilizes around À180
C. Reduce
the flow rate to 8.5 L/min to further reduce temperatures by
about 3
C. Allow the system to cool for an additional 30 min
(see Note 5) When the stage is at cryogenic temperatures, the
chamber vacuum should improve considerably compared to
the room temperature base pressure. On our system, the chamber pressure is around 7 Â 10
À7 mbar when the stage is at
cryogenic temperatures.
Sample Preparation
and Loading
1. Retrieve grid boxes containing the frozen, clipped specimens,
and have them ready in liquid nitrogen.
2. Insert the shuttle into the loading position in the loading
station (Fig. 3a). Fill the loading station with liquid nitrogen
and allow to cool until the liquid is no longer boiling. Refill as
needed. Keep the loading station covered with the standard lid
to minimize atmospheric contamination while cooling.
3. Transfer the grid box containing the grids to the loading
station and unscrew the lid to access the grids.
4. Place one grid into each slot of the shuttle and rotate the grid in
position so that the milling slot is at the top (Figs. 2a and 3e).
5. Secure the grids in the slots by turning the locking screw
(Fig. 3e) at the top of the shuttle. Verify that the grids are
secured by turning the shuttle to the vertical position and check
that the grids remain in place.
6. Flip the shuttle into the vertical transfer position (Fig. 3b) and
replace the standard lid with the transfer lid (Fig. 3d).
7. Press “V” to vent the quickloader airlock (Fig. 3j) and retrieve
the sample transfer arm. Ensure that the transfer arm valve is
closed.
8. Attach the transfer arm to the transfer lid on the loading
station. If present, secure the transfer arm with the locking
clamps (Fig. 3c). Otherwise, securely hold the base of the
transfer arm. Check that the shuttle grabber is in the “open”
position (Fig. 3g).
9. Pump the transfer lid airlock for 35–40 s, then open the transfer arm valve.
64
Vinson Lam and Elizabeth Villa
before filling to minimize ice contamination. Be sure to use
appropriate PPE when handling cryogenic liquids.
10. Fill the heat exchanger tank with liquid nitrogen.
11. Start the temperature logging software to monitor and record
stage and shield temperature.
12. Slowly insert the heat exchanger into the heat exchanger tank
and ensure that it is seated appropriately. Monitor the stage and
shield temperature until it stabilizes around À180
C. Reduce
the flow rate to 8.5 L/min to further reduce temperatures by
about 3
C. Allow the system to cool for an additional 30 min
(see Note 5) When the stage is at cryogenic temperatures, the
chamber vacuum should improve considerably compared to
the room temperature base pressure. On our system, the chamber pressure is around 7 Â 10
À7 mbar when the stage is at
cryogenic temperatures.
Sample Preparation
and Loading
1. Retrieve grid boxes containing the frozen, clipped specimens,
and have them ready in liquid nitrogen.
2. Insert the shuttle into the loading position in the loading
station (Fig. 3a). Fill the loading station with liquid nitrogen
and allow to cool until the liquid is no longer boiling. Refill as
needed. Keep the loading station covered with the standard lid
to minimize atmospheric contamination while cooling.
3. Transfer the grid box containing the grids to the loading
station and unscrew the lid to access the grids.
4. Place one grid into each slot of the shuttle and rotate the grid in
position so that the milling slot is at the top (Figs. 2a and 3e).
5. Secure the grids in the slots by turning the locking screw
(Fig. 3e) at the top of the shuttle. Verify that the grids are
secured by turning the shuttle to the vertical position and check
that the grids remain in place.
6. Flip the shuttle into the vertical transfer position (Fig. 3b) and
replace the standard lid with the transfer lid (Fig. 3d).
7. Press “V” to vent the quickloader airlock (Fig. 3j) and retrieve
the sample transfer arm. Ensure that the transfer arm valve is
closed.
8. Attach the transfer arm to the transfer lid on the loading
station. If present, secure the transfer arm with the locking
clamps (Fig. 3c). Otherwise, securely hold the base of the
transfer arm. Check that the shuttle grabber is in the “open”
position (Fig. 3g).
9. Pump the transfer lid airlock for 35–40 s, then open the transfer arm valve.
64
Vinson Lam and Elizabeth Villa