8. Direct gas flow through the purge tubing (which was previously attached to the front gas port, in step 10 of Subheading
3.3), by blocking flow through the LNP window tubing
(pinching the tubing shut between the thumb and index finger
of the left hand) (see Note 75) while simultaneously blocking
the exhaust outlet in the plastic connector of the LNP nitrogen
withdrawal tubing (using the middle finger of the left hand).
Continue to block both gas exhaust paths until step 11 (see
Note 76).
9. Using the right hand, reach behind the cryomicroscope stage
body and block the rear gas valve with a finger, to pressurize the
chamber gas; after 1–2 s, lift the finger from the rear gas port,
and allow the gas to flow out for approximately 5–10 s. Repeat
this process periodically for 1–2 min, alternatively pressurizing
and releasing the chamber gas (see Note 77).
10. While continuing to block the LNP tubing connector exhaust
outlet and the window tubing, remove the valve opening
connector from the rear gas port by pushing the valve sleeve
toward the stage body until the valve clicks shut.
11. Release the window tubing and stop blocking the LNP tubing
connector exhaust outlet.
12. Set the LNP control to automatic mode (see Note 78).
3.7 Freezing
Experiment
1. Attach the window tubing clip to the stage lid, and adjust the
position of the tubing outlet (see Note 51).
2. Turn on the microscope illuminator (see Note 79).
3. If the cryomicroscope stage body was laterally repositioned
during the experimental setup procedure (see Note 49), then
the stage body should now be re-centered until light can be
observed to pass through the BCS196 silver block aperture (see
Note 50).
4. Configure the microscope for brightfield imaging, and move a
low-power (e.g., 5Â) objective into the microscope’s optical
path (see Note 80).
5. Bring the specimen into focus (relative to the camera live
image, not the eyepieces), and then configure Ko ¨hler illumination (again using the camera live image) (see Note 81).
6. If the BCS196 silver block aperture is not centered in the
camera image, make the necessary fine adjustments to the
cryomicroscope stage body position (see Note 82).
7. Using the microscope’s coarse focus controls, lower the
mechanical stage as far as possible without disturbing the condenser position.
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Jens O. M. Karlsson
3.3), by blocking flow through the LNP window tubing
(pinching the tubing shut between the thumb and index finger
of the left hand) (see Note 75) while simultaneously blocking
the exhaust outlet in the plastic connector of the LNP nitrogen
withdrawal tubing (using the middle finger of the left hand).
Continue to block both gas exhaust paths until step 11 (see
Note 76).
9. Using the right hand, reach behind the cryomicroscope stage
body and block the rear gas valve with a finger, to pressurize the
chamber gas; after 1–2 s, lift the finger from the rear gas port,
and allow the gas to flow out for approximately 5–10 s. Repeat
this process periodically for 1–2 min, alternatively pressurizing
and releasing the chamber gas (see Note 77).
10. While continuing to block the LNP tubing connector exhaust
outlet and the window tubing, remove the valve opening
connector from the rear gas port by pushing the valve sleeve
toward the stage body until the valve clicks shut.
11. Release the window tubing and stop blocking the LNP tubing
connector exhaust outlet.
12. Set the LNP control to automatic mode (see Note 78).
3.7 Freezing
Experiment
1. Attach the window tubing clip to the stage lid, and adjust the
position of the tubing outlet (see Note 51).
2. Turn on the microscope illuminator (see Note 79).
3. If the cryomicroscope stage body was laterally repositioned
during the experimental setup procedure (see Note 49), then
the stage body should now be re-centered until light can be
observed to pass through the BCS196 silver block aperture (see
Note 50).
4. Configure the microscope for brightfield imaging, and move a
low-power (e.g., 5Â) objective into the microscope’s optical
path (see Note 80).
5. Bring the specimen into focus (relative to the camera live
image, not the eyepieces), and then configure Ko ¨hler illumination (again using the camera live image) (see Note 81).
6. If the BCS196 silver block aperture is not centered in the
camera image, make the necessary fine adjustments to the
cryomicroscope stage body position (see Note 82).
7. Using the microscope’s coarse focus controls, lower the
mechanical stage as far as possible without disturbing the condenser position.
236
Jens O. M. Karlsson