sensitive to temperature fluctuations of this magnitude, the
LNP flowrate should be incremented slowly while monitoring
the stage temperature.
75. The metal window tubing clip will be positioned near the end
of the tubing. Try not to disturb the position of the clip relative
to the tubing outlet.
76. There should be an audible hissing sound as the nitrogen gas
flows through the cryomicroscope stage chamber and exits the
rear gas port, flushing out the chamber air.
77. The purpose of this process is to promote gas mixing within the
stage chamber, to more efficiently purge out any moist air. One
may confirm that the chamber is adequately pressurized by
confirming that the stage lid window deflects slightly whenever
the gas valve is blocked (e.g., by observing distortion of light
that is reflected in the lid window). The absence of any noticeable deflection may indicate a leak in the chamber lid and/or
windows, or insufficient nitrogen flow from the pump. It is
very important to wear protective eyewear during the pressurization process, especially while visually observing deflections in
the stage lid window. It should also be noted that the sample
temperature may temporarily fluctuate (by approximately
Æ1
C) during the purging process, due to the perturbation
of coolant flow through the cryomicroscope stage. If the specimen is sensitive to temperature fluctuations of this magnitude,
the duration of blockage of the rear gas port should be
reduced, and the stage temperature should be monitored during the pressurization process and subsequent release.
78. Because the LNP flowrate decreases rapidly when the pump is
switched back to automatic control, the sample temperature
may temporarily rise (by ~1
C). If the specimen is sensitive to
temperature fluctuations of this magnitude, the LNP flowrate
should be manually decreased at a slow to moderate pace,
before switching to automatic control.
79. To minimize the risk of sample damage due to phototoxicity or
excessive heating by the light source, the microscope illumination should remain off (or set to minimal power output) when
the light is not needed. It is also recommended that control
experiments be performed to test for possible influence of
high-intensity illumination on intracellular ice formation
kinetics [23].
80. The objective magnification should be sufficiently low that
the edges of the BCS196 silver block aperture can be seen in
the camera image. If necessary, one may temporarily expand
the field of view by increasing the dimensions of the camera’s
region of interest until the silver block aperture is visible. This
is necessary to make possible adjustment to the centering of the
High-Speed Video Cryomicroscopy
261
LNP flowrate should be incremented slowly while monitoring
the stage temperature.
75. The metal window tubing clip will be positioned near the end
of the tubing. Try not to disturb the position of the clip relative
to the tubing outlet.
76. There should be an audible hissing sound as the nitrogen gas
flows through the cryomicroscope stage chamber and exits the
rear gas port, flushing out the chamber air.
77. The purpose of this process is to promote gas mixing within the
stage chamber, to more efficiently purge out any moist air. One
may confirm that the chamber is adequately pressurized by
confirming that the stage lid window deflects slightly whenever
the gas valve is blocked (e.g., by observing distortion of light
that is reflected in the lid window). The absence of any noticeable deflection may indicate a leak in the chamber lid and/or
windows, or insufficient nitrogen flow from the pump. It is
very important to wear protective eyewear during the pressurization process, especially while visually observing deflections in
the stage lid window. It should also be noted that the sample
temperature may temporarily fluctuate (by approximately
Æ1
C) during the purging process, due to the perturbation
of coolant flow through the cryomicroscope stage. If the specimen is sensitive to temperature fluctuations of this magnitude,
the duration of blockage of the rear gas port should be
reduced, and the stage temperature should be monitored during the pressurization process and subsequent release.
78. Because the LNP flowrate decreases rapidly when the pump is
switched back to automatic control, the sample temperature
may temporarily rise (by ~1
C). If the specimen is sensitive to
temperature fluctuations of this magnitude, the LNP flowrate
should be manually decreased at a slow to moderate pace,
before switching to automatic control.
79. To minimize the risk of sample damage due to phototoxicity or
excessive heating by the light source, the microscope illumination should remain off (or set to minimal power output) when
the light is not needed. It is also recommended that control
experiments be performed to test for possible influence of
high-intensity illumination on intracellular ice formation
kinetics [23].
80. The objective magnification should be sufficiently low that
the edges of the BCS196 silver block aperture can be seen in
the camera image. If necessary, one may temporarily expand
the field of view by increasing the dimensions of the camera’s
region of interest until the silver block aperture is visible. This
is necessary to make possible adjustment to the centering of the
High-Speed Video Cryomicroscopy
261