8. Modify the first row of the temperature profile in the Linksys
software (see Table 1), by changing the entry in the “Time”
column of Ramp 1 from a value of 99,999 to a value of 0, and
then press “Enter” (see Note 83). Confirm that the displayed
stage temperature decreases, as the stage is cooled to the seeding temperature (see Note 41).
9. When the seeding temperature has been reached, use the cryomicroscope stage sample carrier X- and Y-drives to position the
sample edge over the BCS196 seeding cold-spot (see Note 84).
10. Set the LNP control to manual mode, and manually increase
the liquid nitrogen flowrate to its maximum value (by pressing
the upward arrow next to the “Lnp” box in the Temperature
Control Panel, until Lnp ¼ 30). Simultaneously start a stopwatch to time the seeding process.
11. Continuously monitor the cold-spot area through the BCS196
stage lid window. When ice formation is observed at the sample
edge (in the sample region that was resting on the cold-spot),
quickly perform the actions described in steps 12–15 (see Note
85).
12. Stop the stopwatch that was started in step 10 (see Note 86).
13. Switch the LNP control to automatic mode.
14. Turn the cryomicroscope stage X- and Y-drives counterclockwise to move the sample away from the cold-spot, positioning
the sample carrier so that the entire sample rests on the main
(cylindrical) silver block.
15. Advance the execution of the temperature profile (see Table 1),
by changing the entry in the “Time” column of Ramp 2 from a
value of 99,999 to a value of 0, and then press “Enter” (see
Note 87).
16. The microscope and imaging system manipulations that are
described in steps 17–26 must be completed before the end
of the equilibration hold time (see Note 43) that is specified in
Ramp 3 of the temperature profile (e.g., 5 min in the example
in Table 1). If there is insufficient time to complete these
actions, program a longer equilibration hold time in the temperature profile.
17. Move the desired microscope objective into the optical path.
18. Use the microscope focus controls to bring the specimen into
focus in the live camera image.
19. Use the BCS196 sample carrier X- and Y-drives to move a
region of interest in the specimen into the field of view (see
Note 88).
High-Speed Video Cryomicroscopy
237
software (see Table 1), by changing the entry in the “Time”
column of Ramp 1 from a value of 99,999 to a value of 0, and
then press “Enter” (see Note 83). Confirm that the displayed
stage temperature decreases, as the stage is cooled to the seeding temperature (see Note 41).
9. When the seeding temperature has been reached, use the cryomicroscope stage sample carrier X- and Y-drives to position the
sample edge over the BCS196 seeding cold-spot (see Note 84).
10. Set the LNP control to manual mode, and manually increase
the liquid nitrogen flowrate to its maximum value (by pressing
the upward arrow next to the “Lnp” box in the Temperature
Control Panel, until Lnp ¼ 30). Simultaneously start a stopwatch to time the seeding process.
11. Continuously monitor the cold-spot area through the BCS196
stage lid window. When ice formation is observed at the sample
edge (in the sample region that was resting on the cold-spot),
quickly perform the actions described in steps 12–15 (see Note
85).
12. Stop the stopwatch that was started in step 10 (see Note 86).
13. Switch the LNP control to automatic mode.
14. Turn the cryomicroscope stage X- and Y-drives counterclockwise to move the sample away from the cold-spot, positioning
the sample carrier so that the entire sample rests on the main
(cylindrical) silver block.
15. Advance the execution of the temperature profile (see Table 1),
by changing the entry in the “Time” column of Ramp 2 from a
value of 99,999 to a value of 0, and then press “Enter” (see
Note 87).
16. The microscope and imaging system manipulations that are
described in steps 17–26 must be completed before the end
of the equilibration hold time (see Note 43) that is specified in
Ramp 3 of the temperature profile (e.g., 5 min in the example
in Table 1). If there is insufficient time to complete these
actions, program a longer equilibration hold time in the temperature profile.
17. Move the desired microscope objective into the optical path.
18. Use the microscope focus controls to bring the specimen into
focus in the live camera image.
19. Use the BCS196 sample carrier X- and Y-drives to move a
region of interest in the specimen into the field of view (see
Note 88).
High-Speed Video Cryomicroscopy
237