silver block aperture in the microscope’s optical axis, thus
maximizing the effective numerical aperture of the condenser.
81. If the live image is too dark or too small to observe the sample
for focusing purposes, it may be necessary to apply digital
image processing options, such as zoom, gain, or gamma
adjustment, in the camera control software. Conversely, if the
camera image is saturated, temporarily reduce illumination
levels or decrease the camera exposure time to allow focusing.
82. If the BCS196 mounting allows for lateral (XY) movement of
the cryomicroscope stage body relative to the microscope’s
optical axis, use the XY-control on the mechanical stage to
center the silver block aperture in the light path. Alternatively,
the BCS196 mounting hardware may include centering screws
that can be used to align the silver block.
83. Setting a value Time ¼ 0 in an active ramp (i.e., the temperature profile row highlighted in blue in the Linksys software)
that is currently holding at the ramp’s limit temperature will
cause the isothermal hold to terminate, thus advancing the
temperature profile execution to the next ramp in the table.
84. The BCS196 cryomicroscope has a small rectangular silver post
attached to the liquid nitrogen inlet pipe, next to the main
(cylindrical) silver block; this post can be chilled by forcing
nitrogen coolant through the inlet pipe, thus producing a
localized cold-spot that can be used to induce ice formation
in the sample. To induce (i.e., “seed”) ice formation, the edge
of the sample must first be brought to rest over the cold-spot.
With the G16.3 sample carrier, the sample will be in the correct
position for seeding when both the X- and Y-drives have been
turned clockwise as far as possible (however, one should be
careful not to turn these screw drives further when the end of
travel has been reached, because this may result in damage to
the mechanism). When the sample is in the correct position for
seeding, the aperture in the main silver block will be partially
blocked by the edge of the G16.3 carrier.
85. If seeding is successful, the extracellular ice nucleation manifests as a sudden formation of a white cloudy region in the area
of the sample that is located over the cold-spot (and typically
extending a few millimeters beyond the cold-spot). To aid
observation of the seeding process, a small flashlight can be
used to illuminate the cold-spot region through the BCS196
stage lid window.
86. Wait until after the sample has reached the equilibration temperature (after step 15 of Subheading 3.7), and then record
the stopwatch time (which represents the time required to
induce ice formation in the sample). This time should normally
be in the range 10–20 s; seeding times outside this range may
262
Jens O. M. Karlsson
maximizing the effective numerical aperture of the condenser.
81. If the live image is too dark or too small to observe the sample
for focusing purposes, it may be necessary to apply digital
image processing options, such as zoom, gain, or gamma
adjustment, in the camera control software. Conversely, if the
camera image is saturated, temporarily reduce illumination
levels or decrease the camera exposure time to allow focusing.
82. If the BCS196 mounting allows for lateral (XY) movement of
the cryomicroscope stage body relative to the microscope’s
optical axis, use the XY-control on the mechanical stage to
center the silver block aperture in the light path. Alternatively,
the BCS196 mounting hardware may include centering screws
that can be used to align the silver block.
83. Setting a value Time ¼ 0 in an active ramp (i.e., the temperature profile row highlighted in blue in the Linksys software)
that is currently holding at the ramp’s limit temperature will
cause the isothermal hold to terminate, thus advancing the
temperature profile execution to the next ramp in the table.
84. The BCS196 cryomicroscope has a small rectangular silver post
attached to the liquid nitrogen inlet pipe, next to the main
(cylindrical) silver block; this post can be chilled by forcing
nitrogen coolant through the inlet pipe, thus producing a
localized cold-spot that can be used to induce ice formation
in the sample. To induce (i.e., “seed”) ice formation, the edge
of the sample must first be brought to rest over the cold-spot.
With the G16.3 sample carrier, the sample will be in the correct
position for seeding when both the X- and Y-drives have been
turned clockwise as far as possible (however, one should be
careful not to turn these screw drives further when the end of
travel has been reached, because this may result in damage to
the mechanism). When the sample is in the correct position for
seeding, the aperture in the main silver block will be partially
blocked by the edge of the G16.3 carrier.
85. If seeding is successful, the extracellular ice nucleation manifests as a sudden formation of a white cloudy region in the area
of the sample that is located over the cold-spot (and typically
extending a few millimeters beyond the cold-spot). To aid
observation of the seeding process, a small flashlight can be
used to illuminate the cold-spot region through the BCS196
stage lid window.
86. Wait until after the sample has reached the equilibration temperature (after step 15 of Subheading 3.7), and then record
the stopwatch time (which represents the time required to
induce ice formation in the sample). This time should normally
be in the range 10–20 s; seeding times outside this range may
262
Jens O. M. Karlsson