from reaching the camera sensor; this can be achieved by using
the designated microscope control to temporarily divert all
light away from the camera port (and onto the eyepieces).
92. Because it may not be possible to determine whether or not a
cell has frozen unless a high-speed video recording of the
intracellular ice formation event has been captured, it is
extremely important to initiate the video acquisition before
any intracellular crystallization events occur. Thus, if possible,
the video recording should begin at the very start of the rapidfreezing ramp. However, if necessary due to camera memory
limitations, one may delay the start of the high-speed video
recording (i.e., the trigger time) slightly, as long as the stage
temperature remains sufficiently high to preclude any intracellular ice formation prior to the push of the trigger button. If
any of the cells in the field of view have already frozen prior to
the beginning of the acquired video, then the data analysis
methods described in Subheading 3.9 will yield invalid results.
93. During rapid cooling, the sample may go out of focus due to
thermal contraction of the metal components to which the
silver block is mounted. With practice, one may correct such
problems by making small adjustments to the microscope fine
focus during the early stages of freezing. However, it is important to avoid adjusting the microscope focus further when
temperatures have reached a regime in which intracellular ice
formation is likely, because dynamic changes in specimen
appearance in the recorded video will confound detection of
intracellular freezing events.
94. In the Linksys32 software, this is the only available opportunity
to save the recorded temperature data. Thus, it is very important to save the .iml file immediately when prompted. Do not
click “Cancel,” because this will result in permanent loss of
the data.
95. At the end of the video acquisition, the image data exist only in
volatile memory. Thus, the video must be downloaded from
the camera memory and written to the computer hard drive.
The time required to save the video will depend on the amount
of data contained in the memory, the write-speed of the hard
drive, and the data throughput rate of the communication
channels that connect the two devices. It is a good idea to
test (and if possible, optimize) the video download time in
advance, to determine the delay time between successive
experiments.
96. If there is any air gap between the bottom surface of the sample
carrier ring and the top surface of the silver cooling block that
is greater than ~0.1 mm, then the sample carrier may occasionally trap the bottom coverslip of the sample. Thus, to prevent
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Jens O. M. Karlsson
the designated microscope control to temporarily divert all
light away from the camera port (and onto the eyepieces).
92. Because it may not be possible to determine whether or not a
cell has frozen unless a high-speed video recording of the
intracellular ice formation event has been captured, it is
extremely important to initiate the video acquisition before
any intracellular crystallization events occur. Thus, if possible,
the video recording should begin at the very start of the rapidfreezing ramp. However, if necessary due to camera memory
limitations, one may delay the start of the high-speed video
recording (i.e., the trigger time) slightly, as long as the stage
temperature remains sufficiently high to preclude any intracellular ice formation prior to the push of the trigger button. If
any of the cells in the field of view have already frozen prior to
the beginning of the acquired video, then the data analysis
methods described in Subheading 3.9 will yield invalid results.
93. During rapid cooling, the sample may go out of focus due to
thermal contraction of the metal components to which the
silver block is mounted. With practice, one may correct such
problems by making small adjustments to the microscope fine
focus during the early stages of freezing. However, it is important to avoid adjusting the microscope focus further when
temperatures have reached a regime in which intracellular ice
formation is likely, because dynamic changes in specimen
appearance in the recorded video will confound detection of
intracellular freezing events.
94. In the Linksys32 software, this is the only available opportunity
to save the recorded temperature data. Thus, it is very important to save the .iml file immediately when prompted. Do not
click “Cancel,” because this will result in permanent loss of
the data.
95. At the end of the video acquisition, the image data exist only in
volatile memory. Thus, the video must be downloaded from
the camera memory and written to the computer hard drive.
The time required to save the video will depend on the amount
of data contained in the memory, the write-speed of the hard
drive, and the data throughput rate of the communication
channels that connect the two devices. It is a good idea to
test (and if possible, optimize) the video download time in
advance, to determine the delay time between successive
experiments.
96. If there is any air gap between the bottom surface of the sample
carrier ring and the top surface of the silver cooling block that
is greater than ~0.1 mm, then the sample carrier may occasionally trap the bottom coverslip of the sample. Thus, to prevent
264
Jens O. M. Karlsson