[3]. Due to the importance of intracellular crystallization, researchers interested in freezing injury have investigated this phenomenon
by direct microscopic observation of cells exposed to low temperatures, starting with the pioneering work by Go ¨ppert [4]. Such
experimental studies have been facilitated by the development of
cryomicroscopes, optical microscopes that incorporate cooling
technology making possible manipulation of specimen temperature. Whereas the earliest cryomicroscope design was described
over a century ago [5], key advances in cryomicroscopy instrumentation came in the 1970s, with the introduction of automatic
temperature regulation using feedback control [6] and the adoption of then-nascent video recording technology for acquisition of
micrographs at sampling rates up to 60 Hz [7]. The continuous
recording capabilities afforded by video cryomicroscopy are essential for obtaining kinetic information about the stochastic processes
that govern the probability of intracellular freezing. Such data are
valuable for estimating the risk of cryoinjury associated with various
freezing conditions and can therefore be used to guide the development of cryopreservation procedures. Furthermore, quantitative
measurements of intracellular ice formation kinetics are required to
calibrate mathematical models of this damage mechanism, by the
use of curve-fitting techniques to estimate model parameters [8–
12]. In turn, such models make possible computer-aided optimization of complex temperature profiles for minimally damaging freezing and thawing processes [13–15].
Experimental characterization of intracellular ice formation
kinetics is generally done under conditions of rapid cooling (e.g.,
>2 K/s), which minimize the loss of cell water and typically lead to
intracellular freezing in ~100% of the cells observed. The benefit of
preventing freezing-induced cell dehydration is that the estimation
of thermodynamic and kinetic coefficients by model fitting will not
be confounded by changes in the intracellular solute concentrations
[8, 9, 14, 16]: if water transport is negligible, any concentrationdependent model parameters can be treated as constants, while the
relationship between temperature and supercooling can be
described by a simple linear transformation. Unfortunately, conventional cryomicroscopic imaging technology cannot accurately
detect the appearance of ice within rapidly cooled cells. Historically,
investigators have for the most part relied on observations of a
characteristic sudden increase in cytoplasmic opacity as indirect
evidence of intracellular ice formation in the supercooled cell. In
transmitted-light microscopy, this optical phenomenon manifests
as a darkening of the cell, which early researchers described as a
“flashing” [17–19] or “blacking out” [20]. In larger cells (e.g.,
oocytes), the darkening may appear to spread through the cytoplasm at speeds ranging from ~0.1 μm/ms [21] to ~1 μm/ms [18–
22]. Because cells that darken during rapid cooling typically remain
dark for several seconds or longer, it is possible to detect the opacity
222
Jens O. M. Karlsson
by direct microscopic observation of cells exposed to low temperatures, starting with the pioneering work by Go ¨ppert [4]. Such
experimental studies have been facilitated by the development of
cryomicroscopes, optical microscopes that incorporate cooling
technology making possible manipulation of specimen temperature. Whereas the earliest cryomicroscope design was described
over a century ago [5], key advances in cryomicroscopy instrumentation came in the 1970s, with the introduction of automatic
temperature regulation using feedback control [6] and the adoption of then-nascent video recording technology for acquisition of
micrographs at sampling rates up to 60 Hz [7]. The continuous
recording capabilities afforded by video cryomicroscopy are essential for obtaining kinetic information about the stochastic processes
that govern the probability of intracellular freezing. Such data are
valuable for estimating the risk of cryoinjury associated with various
freezing conditions and can therefore be used to guide the development of cryopreservation procedures. Furthermore, quantitative
measurements of intracellular ice formation kinetics are required to
calibrate mathematical models of this damage mechanism, by the
use of curve-fitting techniques to estimate model parameters [8–
12]. In turn, such models make possible computer-aided optimization of complex temperature profiles for minimally damaging freezing and thawing processes [13–15].
Experimental characterization of intracellular ice formation
kinetics is generally done under conditions of rapid cooling (e.g.,
>2 K/s), which minimize the loss of cell water and typically lead to
intracellular freezing in ~100% of the cells observed. The benefit of
preventing freezing-induced cell dehydration is that the estimation
of thermodynamic and kinetic coefficients by model fitting will not
be confounded by changes in the intracellular solute concentrations
[8, 9, 14, 16]: if water transport is negligible, any concentrationdependent model parameters can be treated as constants, while the
relationship between temperature and supercooling can be
described by a simple linear transformation. Unfortunately, conventional cryomicroscopic imaging technology cannot accurately
detect the appearance of ice within rapidly cooled cells. Historically,
investigators have for the most part relied on observations of a
characteristic sudden increase in cytoplasmic opacity as indirect
evidence of intracellular ice formation in the supercooled cell. In
transmitted-light microscopy, this optical phenomenon manifests
as a darkening of the cell, which early researchers described as a
“flashing” [17–19] or “blacking out” [20]. In larger cells (e.g.,
oocytes), the darkening may appear to spread through the cytoplasm at speeds ranging from ~0.1 μm/ms [21] to ~1 μm/ms [18–
22]. Because cells that darken during rapid cooling typically remain
dark for several seconds or longer, it is possible to detect the opacity
222
Jens O. M. Karlsson