indicate that the seeding temperature was not properly
selected. When running multiple experiments with replicate
samples, outliers in the seeding time may signify a problem
with the sample (e.g., dehydration during sample preparation)
or with the cryomicroscope stage (e.g., impeded heat transfer
due to scratches or foreign material on the silver block surface,
or blockage of liquid nitrogen flow).
87. This will advance the execution of the temperature profile to
Ramp 3 in Table 1, thus bringing the sample to the postseeding equilibration temperature. Steps 14 and 15 (Subheading 3.7) must be completed in quick succession, so that the ice
patch formed during seeding does not melt (which may happen
if the seeding temperature is above the solution melting temperature) or grow too rapidly (which may happen if the seeding
temperature is below the equilibration temperature). As a general guideline, the size of the ice patch at the sample edge
should not change by more than Æ50% before the sample has
reached the equilibration hold temperature.
88. During cooling, the sample may drift laterally, due to thermal
contraction of the metals in the BCS196 stage. Thus, when
selecting a field of view, allow for a margin between the cells of
interest and the image edge.
89. All adjustments should be made with reference to the camera
live image (not the microscope eyepieces). For highmagnification objectives, if the image field of view is too small
to contain the blade edges of the stopped-down field diaphragm, one may temporarily expand the field of view by
increasing the dimensions of the camera’s region of interest.
Only minor adjustments to the condenser focus and field
diaphragm should be necessary, because the microscope was
already pre-configured for Ko ¨hler illumination in step 5 of
Subheading 3.7.
90. The intracellular ice crystals (which have not yet formed) do
not have a high degree of contrast. Thus, the condenser diaphragm aperture typically needs to be significantly restricted in
order to detect intracellular ice formation. Some iteration may
be necessary to select the best aperture size, because the contrast of the intracellular ice crystals can only be checked during
slow-motion playback of the high-speed video that is recorded
during freezing.
91. In the Vision Research Phantom cameras, this is done using the
“Current Session Reference” function. For optimal image
quality, the camera should be recalibrated before each video
recording, because the black levels are sensitive to changes in
temperature as the camera warms up during continuous operation. Black level calibration requires that light be prevented
High-Speed Video Cryomicroscopy
263
selected. When running multiple experiments with replicate
samples, outliers in the seeding time may signify a problem
with the sample (e.g., dehydration during sample preparation)
or with the cryomicroscope stage (e.g., impeded heat transfer
due to scratches or foreign material on the silver block surface,
or blockage of liquid nitrogen flow).
87. This will advance the execution of the temperature profile to
Ramp 3 in Table 1, thus bringing the sample to the postseeding equilibration temperature. Steps 14 and 15 (Subheading 3.7) must be completed in quick succession, so that the ice
patch formed during seeding does not melt (which may happen
if the seeding temperature is above the solution melting temperature) or grow too rapidly (which may happen if the seeding
temperature is below the equilibration temperature). As a general guideline, the size of the ice patch at the sample edge
should not change by more than Æ50% before the sample has
reached the equilibration hold temperature.
88. During cooling, the sample may drift laterally, due to thermal
contraction of the metals in the BCS196 stage. Thus, when
selecting a field of view, allow for a margin between the cells of
interest and the image edge.
89. All adjustments should be made with reference to the camera
live image (not the microscope eyepieces). For highmagnification objectives, if the image field of view is too small
to contain the blade edges of the stopped-down field diaphragm, one may temporarily expand the field of view by
increasing the dimensions of the camera’s region of interest.
Only minor adjustments to the condenser focus and field
diaphragm should be necessary, because the microscope was
already pre-configured for Ko ¨hler illumination in step 5 of
Subheading 3.7.
90. The intracellular ice crystals (which have not yet formed) do
not have a high degree of contrast. Thus, the condenser diaphragm aperture typically needs to be significantly restricted in
order to detect intracellular ice formation. Some iteration may
be necessary to select the best aperture size, because the contrast of the intracellular ice crystals can only be checked during
slow-motion playback of the high-speed video that is recorded
during freezing.
91. In the Vision Research Phantom cameras, this is done using the
“Current Session Reference” function. For optimal image
quality, the camera should be recalibrated before each video
recording, because the black levels are sensitive to changes in
temperature as the camera warms up during continuous operation. Black level calibration requires that light be prevented
High-Speed Video Cryomicroscopy
263