20. Optionally, acquire fluorescent images (e.g., to identify live and
dead cells in the field of view), and then switch the microscope
optics back to brightfield illumination.
21. Make any necessary adjustments to the fine focus and to the
Ko ¨hler illumination (see Note 89).
22. Adjust the condenser iris to achieve an adequate balance
between image contrast, brightness, and optical resolution
(see Note 90).
23. Make any necessary adjustments to the camera exposure, image
size, and other settings in the camera control software.
24. Calibrate the camera black level (see Note 91).
25. Start capturing video into the camera’s circular memory buffer.
The camera should now be waiting for the trigger signal.
26. Pick up the trigger switch, and be ready to push the trigger
button during the next step.
27. Simultaneously monitor the Linksys stage temperature display
and status indicators, as well as the camera live image. When
the Linksys software automatically advances the temperature
profile to the rapid-cooling ramp (Ramp 4 in Table 1), push the
trigger button (see Notes 92 and 93).
28. When the high-speed video recording is completed, click the
blue square button in the Linksys software Data Capture toolbar to stop recording temperature data. When prompted,
specify a directory and name for the Linksys data (.iml) file,
and click “Save” (see Note 94).
29. Turn off the microscope illumination.
30. Save the acquired video onto the computer hard drive. If any
still images were captured during the experiment, save these as
well (see Note 95).
31. Set the LNP control to manual mode, and set the flowrate to
zero (by pressing the downward arrow next to the “Lnp” box
in the Temperature Control Panel) (see Note 48).
32. Maximize clearance between the BCS196 lid and the microscope objectives by using the coarse focus controls to lower the
stage (see Notes 49 and 50).
33. Detach the window tubing clip from the BCS196 stage lid, and
then unscrew and remove the lid. Set the lid upside-down onto
a clean, dry surface.
34. Use the BCS196 stage X- and Y-drives to center the sample
carrier on the silver block in the cryomicroscope chamber. Use
vacuum tweezers to remove the previous sample from the silver
block (see Note 96).
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Jens O. M. Karlsson
dead cells in the field of view), and then switch the microscope
optics back to brightfield illumination.
21. Make any necessary adjustments to the fine focus and to the
Ko ¨hler illumination (see Note 89).
22. Adjust the condenser iris to achieve an adequate balance
between image contrast, brightness, and optical resolution
(see Note 90).
23. Make any necessary adjustments to the camera exposure, image
size, and other settings in the camera control software.
24. Calibrate the camera black level (see Note 91).
25. Start capturing video into the camera’s circular memory buffer.
The camera should now be waiting for the trigger signal.
26. Pick up the trigger switch, and be ready to push the trigger
button during the next step.
27. Simultaneously monitor the Linksys stage temperature display
and status indicators, as well as the camera live image. When
the Linksys software automatically advances the temperature
profile to the rapid-cooling ramp (Ramp 4 in Table 1), push the
trigger button (see Notes 92 and 93).
28. When the high-speed video recording is completed, click the
blue square button in the Linksys software Data Capture toolbar to stop recording temperature data. When prompted,
specify a directory and name for the Linksys data (.iml) file,
and click “Save” (see Note 94).
29. Turn off the microscope illumination.
30. Save the acquired video onto the computer hard drive. If any
still images were captured during the experiment, save these as
well (see Note 95).
31. Set the LNP control to manual mode, and set the flowrate to
zero (by pressing the downward arrow next to the “Lnp” box
in the Temperature Control Panel) (see Note 48).
32. Maximize clearance between the BCS196 lid and the microscope objectives by using the coarse focus controls to lower the
stage (see Notes 49 and 50).
33. Detach the window tubing clip from the BCS196 stage lid, and
then unscrew and remove the lid. Set the lid upside-down onto
a clean, dry surface.
34. Use the BCS196 stage X- and Y-drives to center the sample
carrier on the silver block in the cryomicroscope chamber. Use
vacuum tweezers to remove the previous sample from the silver
block (see Note 96).
238
Jens O. M. Karlsson