concentrated liquid in the unfrozen fraction. The equilibration
hold also needs to be sufficiently long to allow time for selecting a field of view, adjusting the microscope optics (e.g., fine
focus, Ko ¨hler illumination, and contrast enhancement), and
any other pre-freezing experimental procedures (such as acquisition of fluorescence images). The length of the equilibration
hold will also affect the size and shape of the extracellular ice
crystals (due to annealing) and, if excessively long, may cause
some degradation of cell quality due to the action of “solution
effects” mechanisms. Because both of these factors (geometry
of ice-cell interactions and exposure to the unfrozen solution)
may affect the subsequent intracellular ice formation kinetics, it
is important to keep the equilibration hold time consistent
from experiment to experiment. In the example in Table 1,
the hold time has been set to 5 min. Note that the Linksys32
software allows for the unit of time to be configured as either
minutes or seconds. The time field in the Temperature Control
Panel will be labeled “Hold mins” if the unit of time is minutes,
or “Hold secs” if the time is measured in seconds; in the latter
case, the temperature profile Ramp 3 in Table 1 should be
modified so that Time ¼ 300 in order to achieve a 5-min
equilibration hold.
44. The rate of cooling (in
C/min) for the main freezing ramp is
an important experimental parameter. For measurement of
intracellular ice formation kinetics, the cooling rate is typically
set to the fastest controlled rate achievable by the cryomicroscope system. This minimizes the amount of cell dehydration
during freezing, which significantly simplifies data interpretation, by allowing the analyses of intracellular ice formation and
water transport to be decoupled (because, for negligible levels
of water loss, the chemical composition of the intracellular
liquid can be assumed to be constant during freezing). Alternatively, the cooling rate may be varied as part of the experimental design (e.g., to estimate the critical cooling rate, at
which the cumulative incidence of intracellular ice formation
is 50%). In some cases (especially when working with thicker
samples, which can develop vertical temperature gradients during rapid cooling), one may also choose to limit the cooling
rate in order to reduce temperature measurement errors.
45. The experiment end temperature should be selected so that
intracellular ice formation has been completed before the end
of the freezing ramp. For mammalian cells frozen rapidly (i.e.,
with negligible dehydration) in isotonic solution without cryoprotectants, the lowest expected intracellular freezing point is
approximately À40
C (corresponding to ice formation by
homogeneous nucleation); the theoretical minimum will be
lower if the freezing point is depressed by the use of
High-Speed Video Cryomicroscopy
253
hold also needs to be sufficiently long to allow time for selecting a field of view, adjusting the microscope optics (e.g., fine
focus, Ko ¨hler illumination, and contrast enhancement), and
any other pre-freezing experimental procedures (such as acquisition of fluorescence images). The length of the equilibration
hold will also affect the size and shape of the extracellular ice
crystals (due to annealing) and, if excessively long, may cause
some degradation of cell quality due to the action of “solution
effects” mechanisms. Because both of these factors (geometry
of ice-cell interactions and exposure to the unfrozen solution)
may affect the subsequent intracellular ice formation kinetics, it
is important to keep the equilibration hold time consistent
from experiment to experiment. In the example in Table 1,
the hold time has been set to 5 min. Note that the Linksys32
software allows for the unit of time to be configured as either
minutes or seconds. The time field in the Temperature Control
Panel will be labeled “Hold mins” if the unit of time is minutes,
or “Hold secs” if the time is measured in seconds; in the latter
case, the temperature profile Ramp 3 in Table 1 should be
modified so that Time ¼ 300 in order to achieve a 5-min
equilibration hold.
44. The rate of cooling (in
C/min) for the main freezing ramp is
an important experimental parameter. For measurement of
intracellular ice formation kinetics, the cooling rate is typically
set to the fastest controlled rate achievable by the cryomicroscope system. This minimizes the amount of cell dehydration
during freezing, which significantly simplifies data interpretation, by allowing the analyses of intracellular ice formation and
water transport to be decoupled (because, for negligible levels
of water loss, the chemical composition of the intracellular
liquid can be assumed to be constant during freezing). Alternatively, the cooling rate may be varied as part of the experimental design (e.g., to estimate the critical cooling rate, at
which the cumulative incidence of intracellular ice formation
is 50%). In some cases (especially when working with thicker
samples, which can develop vertical temperature gradients during rapid cooling), one may also choose to limit the cooling
rate in order to reduce temperature measurement errors.
45. The experiment end temperature should be selected so that
intracellular ice formation has been completed before the end
of the freezing ramp. For mammalian cells frozen rapidly (i.e.,
with negligible dehydration) in isotonic solution without cryoprotectants, the lowest expected intracellular freezing point is
approximately À40
C (corresponding to ice formation by
homogeneous nucleation); the theoretical minimum will be
lower if the freezing point is depressed by the use of
High-Speed Video Cryomicroscopy
253