in Subheadding 3.9, only the video images acquired after the
trigger event are used.
58. If the recording length is significantly shorter than the duration
of the cooling portion of the ramp, then the cryomicroscopy
videos will be prematurely truncated, which may result in
missing intracellular ice formation events. One may estimate
the temperature at which the video recording will terminate by
expressing the computed post-trigger recording duration in
minutes and multiplying by the cooling rate from Ramp 4 in
Table 1 (in
C/min) and then subtracting this product from
the ramp start temperature (i.e., the “Limit” value in Ramp
3 of Table 1).
59. Alternatively, if the recording end time occurs too late, one may
reduce the number of post-trigger frames. In particular, to
establish an appropriate number of post-trigger frames, subtract 1 s from the calculated cooling ramp duration (including
the duration of any isothermal hold at the ramp end temperature), and then multiply the result by the image acquisition
rate. Note that any video frames that are acquired before the
trigger will not be used in the data analysis. If less than half of
the total number of frames available for recording will be
acquired after the trigger, then a more suitable approach to
reducing the recording duration is to partition the camera
memory.
60. To save Vision Research Phantom camera setup file (.stp format), complete the following steps: First, in the PCC software,
expand the “Advanced Settings” section of the “Live” panel,
and click the “Save Settings” button; conversely, in the legacy
camera control application, click the “Save. . .” button in the
“Setup and Recording” window. Second, if using the PCC
software, set the file type selection to “All (∗.∗)”; in the legacy
application, set the file type to “Setup files (∗.STP).” Third,
specify the desired location and name of the setup file, including the .stp extension (the file will not be properly saved if the .
stp extension is not explicitly appended to the specified file
name), and click the “Save” button.
61. To clean off grease residue from the external surfaces of the
syringe, isopropyl alcohol works well.
62. Dispensing needles in the range 16–20 G are recommended.
With the larger bores, less pressure is needed to dispense
grease, whereas the smaller bores offer finer control of the
amount dispensed.
63. If the dispenser has not been used for some time, the dispensing tip should be cleared by pushing some grease through the
syringe, before use in sample preparation.
258
Jens O. M. Karlsson
trigger event are used.
58. If the recording length is significantly shorter than the duration
of the cooling portion of the ramp, then the cryomicroscopy
videos will be prematurely truncated, which may result in
missing intracellular ice formation events. One may estimate
the temperature at which the video recording will terminate by
expressing the computed post-trigger recording duration in
minutes and multiplying by the cooling rate from Ramp 4 in
Table 1 (in
C/min) and then subtracting this product from
the ramp start temperature (i.e., the “Limit” value in Ramp
3 of Table 1).
59. Alternatively, if the recording end time occurs too late, one may
reduce the number of post-trigger frames. In particular, to
establish an appropriate number of post-trigger frames, subtract 1 s from the calculated cooling ramp duration (including
the duration of any isothermal hold at the ramp end temperature), and then multiply the result by the image acquisition
rate. Note that any video frames that are acquired before the
trigger will not be used in the data analysis. If less than half of
the total number of frames available for recording will be
acquired after the trigger, then a more suitable approach to
reducing the recording duration is to partition the camera
memory.
60. To save Vision Research Phantom camera setup file (.stp format), complete the following steps: First, in the PCC software,
expand the “Advanced Settings” section of the “Live” panel,
and click the “Save Settings” button; conversely, in the legacy
camera control application, click the “Save. . .” button in the
“Setup and Recording” window. Second, if using the PCC
software, set the file type selection to “All (∗.∗)”; in the legacy
application, set the file type to “Setup files (∗.STP).” Third,
specify the desired location and name of the setup file, including the .stp extension (the file will not be properly saved if the .
stp extension is not explicitly appended to the specified file
name), and click the “Save” button.
61. To clean off grease residue from the external surfaces of the
syringe, isopropyl alcohol works well.
62. Dispensing needles in the range 16–20 G are recommended.
With the larger bores, less pressure is needed to dispense
grease, whereas the smaller bores offer finer control of the
amount dispensed.
63. If the dispenser has not been used for some time, the dispensing tip should be cleared by pushing some grease through the
syringe, before use in sample preparation.
258
Jens O. M. Karlsson