20. After the appropriate length of window tubing has been pulled
through the window tubing clip, carefully detach the clip from
the cryomicroscope stage lid (without altering the length of
tubing that has been threaded through the hole in the
metal clip).
21. Unscrew and remove the BCS196 stage lid, and set it upsidedown onto a clean, dry surface.
22. Use the BCS196 stage X- and Y-drives to center the sample
carrier on the silver block in the cryomicroscope chamber.
23. Visually inspect the silver block to ensure that it is clean and
dry. Dust particles can be removed using a bulb air blower.
24. Replace the cryomicroscope stage lid to minimize dust ingress
during sample preparation. It is not necessary to tighten the lid
completely.
3.4 Initial
Configuration
of High-Speed Imaging
System
1. Launch the camera control software and navigate to the video
acquisition settings interface (see Note 52).
2. If a camera setup file has previously been saved (see step 11
below), then load the saved settings into the software (see Note
53), and proceed to the sample preparation procedure (Subheading 3.5). Otherwise, complete the camera configuration
procedure described below.
3. Set the camera sensor’s active region, or region of interest
(ROI), by selecting the desired image dimensions (in pixels)
from the “Resolution” drop-down menu, or by typing in a
custom value and pressing “Enter” (see Note 54).
4. Set the camera exposure time to 100 μs (see Note 55).
5. Set the camera image acquisition rate (“Sample rate” in the
Phantom Camera Control software) to a value in the range
2000–8000 fps (see Note 56).
6. Configure the video recording termination time by specifying
the number of frames to be acquired after the trigger event is
detected. Start by placing the trigger position at the beginning
of the recorded image range (i.e., make all frames post-trigger)
(see Note 57).
7. Determine the post-trigger recording time by dividing the
number of post-trigger frames by the camera’s image
acquisition rate.
8. Confirm that the available post-trigger recording duration is
approximately equal to the duration of the rapid-cooling ramp
in the temperature profile (Ramp 4 in Table 1), which is computed by taking the difference between the ramp’s start and
end temperatures, and then dividing this difference by the
cooling rate. If the post-trigger recording length is significantly
232
Jens O. M. Karlsson
through the window tubing clip, carefully detach the clip from
the cryomicroscope stage lid (without altering the length of
tubing that has been threaded through the hole in the
metal clip).
21. Unscrew and remove the BCS196 stage lid, and set it upsidedown onto a clean, dry surface.
22. Use the BCS196 stage X- and Y-drives to center the sample
carrier on the silver block in the cryomicroscope chamber.
23. Visually inspect the silver block to ensure that it is clean and
dry. Dust particles can be removed using a bulb air blower.
24. Replace the cryomicroscope stage lid to minimize dust ingress
during sample preparation. It is not necessary to tighten the lid
completely.
3.4 Initial
Configuration
of High-Speed Imaging
System
1. Launch the camera control software and navigate to the video
acquisition settings interface (see Note 52).
2. If a camera setup file has previously been saved (see step 11
below), then load the saved settings into the software (see Note
53), and proceed to the sample preparation procedure (Subheading 3.5). Otherwise, complete the camera configuration
procedure described below.
3. Set the camera sensor’s active region, or region of interest
(ROI), by selecting the desired image dimensions (in pixels)
from the “Resolution” drop-down menu, or by typing in a
custom value and pressing “Enter” (see Note 54).
4. Set the camera exposure time to 100 μs (see Note 55).
5. Set the camera image acquisition rate (“Sample rate” in the
Phantom Camera Control software) to a value in the range
2000–8000 fps (see Note 56).
6. Configure the video recording termination time by specifying
the number of frames to be acquired after the trigger event is
detected. Start by placing the trigger position at the beginning
of the recorded image range (i.e., make all frames post-trigger)
(see Note 57).
7. Determine the post-trigger recording time by dividing the
number of post-trigger frames by the camera’s image
acquisition rate.
8. Confirm that the available post-trigger recording duration is
approximately equal to the duration of the rapid-cooling ramp
in the temperature profile (Ramp 4 in Table 1), which is computed by taking the difference between the ramp’s start and
end temperatures, and then dividing this difference by the
cooling rate. If the post-trigger recording length is significantly
232
Jens O. M. Karlsson