body relative to the microscope, then the BCS196 should be
moved as far toward the front of the microscope as possible.
50. Note that for certain combinations of cryomicroscope mounting hardware designs and microscope condenser optics, it is
possible for the cryomicroscope’s mounting adapter to make
contact with the condenser top lens if the BCS196 stage body
is moved laterally (in the XY plane) while the condenser focus
position has been set for Ko ¨hler illumination. Thus, to avoid
damage in such cases, one should make a habit of always
lowering the condenser before making any changes to the
position of the cryomicroscope stage body.
51. The end of the window tubing should touch the edge of the
stage lid window. It may be necessary to position the tubing
closer to the window center (if frost forms in the light path
during freezing experiments) or further away from the window
edge (if the tubing interferes with objectives that have shorter
working distance).
52. In the modern (post-2010) versions of Vision Research Phantom Camera Control (PCC) software, click the “Live” panel
tab, and expand the “Cine Settings” section. In the legacy
version of the Vision Research Phantom Camera Control application, select “Setup and Recording. . .” under the
Acquisition menu.
53. To load a previously saved camera setup file for Vision Research
Phantom cameras (.stp format), complete the following steps:
First, in the PCC software, expand the “Advanced Settings”
section of the “Live” panel, and click the “Load Settings”
button; conversely, in the legacy camera control application,
click the “Open. . .” button in the “Setup and Recording”
window. Second, if using the PCC software, set the file type
selection to “All (∗.∗)”; in the legacy application, ensure the
file type is set to “Setup files (∗.STP,∗.STG).” Third, select
the desired setup file (.stp extension) and click the “Open”
button.
54. The ROI dimensions should be selected to accommodate the
size of the field under observation, including a margin to allow
for sample drift during cooling (e.g., due to thermal contraction of the coolant pipes); for example, on a Linkam Scientific
Instruments BCS196 cryomicroscope stage, the average drift is
on the order of 1 μm/K. The larger the ROI, the more data can
be collected for each freezing run (because of the larger number of cells in the field of view). The maximum ROI size is
determined by the physical dimensions of the camera sensor. In
addition, because the view of the specimen is restricted by the
1.3-mm diameter circular aperture in the silver block of the
cryomicroscope stage, any part of the image that is obstructed
High-Speed Video Cryomicroscopy
255
moved as far toward the front of the microscope as possible.
50. Note that for certain combinations of cryomicroscope mounting hardware designs and microscope condenser optics, it is
possible for the cryomicroscope’s mounting adapter to make
contact with the condenser top lens if the BCS196 stage body
is moved laterally (in the XY plane) while the condenser focus
position has been set for Ko ¨hler illumination. Thus, to avoid
damage in such cases, one should make a habit of always
lowering the condenser before making any changes to the
position of the cryomicroscope stage body.
51. The end of the window tubing should touch the edge of the
stage lid window. It may be necessary to position the tubing
closer to the window center (if frost forms in the light path
during freezing experiments) or further away from the window
edge (if the tubing interferes with objectives that have shorter
working distance).
52. In the modern (post-2010) versions of Vision Research Phantom Camera Control (PCC) software, click the “Live” panel
tab, and expand the “Cine Settings” section. In the legacy
version of the Vision Research Phantom Camera Control application, select “Setup and Recording. . .” under the
Acquisition menu.
53. To load a previously saved camera setup file for Vision Research
Phantom cameras (.stp format), complete the following steps:
First, in the PCC software, expand the “Advanced Settings”
section of the “Live” panel, and click the “Load Settings”
button; conversely, in the legacy camera control application,
click the “Open. . .” button in the “Setup and Recording”
window. Second, if using the PCC software, set the file type
selection to “All (∗.∗)”; in the legacy application, ensure the
file type is set to “Setup files (∗.STP,∗.STG).” Third, select
the desired setup file (.stp extension) and click the “Open”
button.
54. The ROI dimensions should be selected to accommodate the
size of the field under observation, including a margin to allow
for sample drift during cooling (e.g., due to thermal contraction of the coolant pipes); for example, on a Linkam Scientific
Instruments BCS196 cryomicroscope stage, the average drift is
on the order of 1 μm/K. The larger the ROI, the more data can
be collected for each freezing run (because of the larger number of cells in the field of view). The maximum ROI size is
determined by the physical dimensions of the camera sensor. In
addition, because the view of the specimen is restricted by the
1.3-mm diameter circular aperture in the silver block of the
cryomicroscope stage, any part of the image that is obstructed
High-Speed Video Cryomicroscopy
255