forces from being transmitted to the sample carrier when
lifting the sample out, it is advisable to first check whether
the sample is stuck. This can be done by gently nudging the
sample horizontally, to check whether it slides freely before
making contact with the inner edge of the sample carrier ring
(there should be a space of approximately 0.3 mm between
the sample coverslip edge and the sample carrier ring).
97. To load a previously saved profile file, complete the following steps: First, stop the profile execution by clicking the
blue square button in the Temperature Control toolbar.
Second, click on any cell in the “Delay” column of the
temperature profile table. Third, go to the “File” menu
and select “Open” and then “Temperature Profile”; select
the desired profile file (.pro extension) and click the “Open”
button. Finally, restart profile execution as soon as possible,
by clicking the blue triangle in the Temperature Control
toolbar. Ensure that the stage temperature is holding at the
desired sample loading temperature.
98. To reduce wear, employ a twisting motion to ease the tubing
connectors off the metal pipes, instead of pulling them off.
99. Be careful not to twist or bend the black capillary tubing. Be
mindful of small amounts of liquid nitrogen that may be
ejected from the end of the capillary tube, due to pressure
buildup in the dewar.
100. In Vision Research Phantom cameras, the first post-trigger
frame always corresponds to frame #0.
101. It is important to avoid introducing bias: thus, the criteria for
removing cells from further analysis should be independent of
the probability of ice formation in the cells. For example, even
if a cell doesn’t disappear from the field of view until after its
intracellular freezing event, it cannot be included in the tally.
In addition, one should also omit cells that are not yet visible
at the video trigger time, but that drift into the field of view
during the course of the freezing ramp. It is recommended
that attempts be made during the cryomicroscope experiment
to minimize the probability of data censoring (e.g., by selecting a field of view in which there is a sufficient margin between
the cells and the edges of the field to account for drift).
Furthermore, it should be noted that the Nelson-Aalen algorithm does allow for a more flexible approach to data censoring, but such variations of the data analysis technique are
outside the scope of this chapter.
102. If the temporal resolution of the acquired video is sufficiently
fine (e.g., sampling intervals on the order 0.5 ms or better),
then the majority of intracellular ice formation events will
manifest as a solitary wavefront that travels through the cell
High-Speed Video Cryomicroscopy
265
lifting the sample out, it is advisable to first check whether
the sample is stuck. This can be done by gently nudging the
sample horizontally, to check whether it slides freely before
making contact with the inner edge of the sample carrier ring
(there should be a space of approximately 0.3 mm between
the sample coverslip edge and the sample carrier ring).
97. To load a previously saved profile file, complete the following steps: First, stop the profile execution by clicking the
blue square button in the Temperature Control toolbar.
Second, click on any cell in the “Delay” column of the
temperature profile table. Third, go to the “File” menu
and select “Open” and then “Temperature Profile”; select
the desired profile file (.pro extension) and click the “Open”
button. Finally, restart profile execution as soon as possible,
by clicking the blue triangle in the Temperature Control
toolbar. Ensure that the stage temperature is holding at the
desired sample loading temperature.
98. To reduce wear, employ a twisting motion to ease the tubing
connectors off the metal pipes, instead of pulling them off.
99. Be careful not to twist or bend the black capillary tubing. Be
mindful of small amounts of liquid nitrogen that may be
ejected from the end of the capillary tube, due to pressure
buildup in the dewar.
100. In Vision Research Phantom cameras, the first post-trigger
frame always corresponds to frame #0.
101. It is important to avoid introducing bias: thus, the criteria for
removing cells from further analysis should be independent of
the probability of ice formation in the cells. For example, even
if a cell doesn’t disappear from the field of view until after its
intracellular freezing event, it cannot be included in the tally.
In addition, one should also omit cells that are not yet visible
at the video trigger time, but that drift into the field of view
during the course of the freezing ramp. It is recommended
that attempts be made during the cryomicroscope experiment
to minimize the probability of data censoring (e.g., by selecting a field of view in which there is a sufficient margin between
the cells and the edges of the field to account for drift).
Furthermore, it should be noted that the Nelson-Aalen algorithm does allow for a more flexible approach to data censoring, but such variations of the data analysis technique are
outside the scope of this chapter.
102. If the temporal resolution of the acquired video is sufficiently
fine (e.g., sampling intervals on the order 0.5 ms or better),
then the majority of intracellular ice formation events will
manifest as a solitary wavefront that travels through the cell
High-Speed Video Cryomicroscopy
265