seeding temperature is too high, the ice formed during the
seeding process may melt before the temperature profile can be
advanced to the next ramp (the equilibration hold). Conversely, if the seeding temperature is too low, rapid growth of
extracellular ice throughout the specimen may cause damage to
some of the cells. As a general guideline, the seeding temperature should be selected so that the seeding process results in
localized ice formation (not extending more than a few millimeters beyond the seeding cold-spot) within 10–20 s; moreover, the size of the resulting ice patch should not change by
more than Æ50% in the time it take for the sample to reach the
equilibration hold temperature (Ramp 3 in Table 1). When
experimenting with different seeding temperatures, it is helpful
to directly observe the formation and subsequent changes to
the localized ice patch (which appears as a white cloudy area),
by using a small flashlight to illuminate the sample edge
through the BCS196 stage lid window.
42. The equilibration temperature is the temperature at which the
sample will be held after seeding, to allow the extracellular ice
to propagate throughout the sample, until it reaches equilibrium with the unfrozen solution. This temperature must be
below the melting point of the solution. The equilibration
temperature should be chosen according to the needs of the
experiment. If the equilibration temperature is low, then the
unfrozen liquid fraction will be lower (possibly causing cell
crowding or excessive deformation if too low), and the initial
water content within the cell will be reduced (which will
depress intracellular freezing temperatures). If the equilibration temperature is high (approaching the melting temperature), then cell dehydration will be minimal, but the volume
fraction of ice in the sample may be too low. If the amount of
extracellular ice in the sample is so low that many cells are
initially not in contact with ice, then the experimental results
may be more difficult to interpret (because the probability of
intracellular ice formation is known to be affected by contact
with extracellular ice crystals). Moreover, if rapid freezing
(Ramp 4 in Table 1) is initiated while the unfrozen liquid
fraction is large, then fine dendritic ice crystals will fill the
unfrozen spaces in the early stages of the freezing ramp,
which may cause the view of the cells to be obscured.
43. The equilibration hold time is the time at which the sample will
be held at the equilibration hold temperature before rapid
freezing (Ramp 4 in Table 1) is initiated. To facilitate interpretation of experimental results, the hold time should be sufficiently long to allow the ice field to advance through the area of
the sample to be observed during freezing and to allow cells in
this region to reach osmotic equilibrium with the freeze252
Jens O. M. Karlsson
seeding process may melt before the temperature profile can be
advanced to the next ramp (the equilibration hold). Conversely, if the seeding temperature is too low, rapid growth of
extracellular ice throughout the specimen may cause damage to
some of the cells. As a general guideline, the seeding temperature should be selected so that the seeding process results in
localized ice formation (not extending more than a few millimeters beyond the seeding cold-spot) within 10–20 s; moreover, the size of the resulting ice patch should not change by
more than Æ50% in the time it take for the sample to reach the
equilibration hold temperature (Ramp 3 in Table 1). When
experimenting with different seeding temperatures, it is helpful
to directly observe the formation and subsequent changes to
the localized ice patch (which appears as a white cloudy area),
by using a small flashlight to illuminate the sample edge
through the BCS196 stage lid window.
42. The equilibration temperature is the temperature at which the
sample will be held after seeding, to allow the extracellular ice
to propagate throughout the sample, until it reaches equilibrium with the unfrozen solution. This temperature must be
below the melting point of the solution. The equilibration
temperature should be chosen according to the needs of the
experiment. If the equilibration temperature is low, then the
unfrozen liquid fraction will be lower (possibly causing cell
crowding or excessive deformation if too low), and the initial
water content within the cell will be reduced (which will
depress intracellular freezing temperatures). If the equilibration temperature is high (approaching the melting temperature), then cell dehydration will be minimal, but the volume
fraction of ice in the sample may be too low. If the amount of
extracellular ice in the sample is so low that many cells are
initially not in contact with ice, then the experimental results
may be more difficult to interpret (because the probability of
intracellular ice formation is known to be affected by contact
with extracellular ice crystals). Moreover, if rapid freezing
(Ramp 4 in Table 1) is initiated while the unfrozen liquid
fraction is large, then fine dendritic ice crystals will fill the
unfrozen spaces in the early stages of the freezing ramp,
which may cause the view of the cells to be obscured.
43. The equilibration hold time is the time at which the sample will
be held at the equilibration hold temperature before rapid
freezing (Ramp 4 in Table 1) is initiated. To facilitate interpretation of experimental results, the hold time should be sufficiently long to allow the ice field to advance through the area of
the sample to be observed during freezing and to allow cells in
this region to reach osmotic equilibrium with the freeze252
Jens O. M. Karlsson