Chapter 8
High-Speed Video Cryomicroscopy for Measurement
of Intracellular Ice Formation Kinetics
Jens O. M. Karlsson
Abstract
Quantitative information about the kinetics and cumulative probability of intracellular ice formation is
necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues.
Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity
changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the
formation and growth of intracellular ice crystals can be accurately detected using temporally resolved
imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the
setup and operation of a high-speed video cryomicroscope system are described, including protocols for
imaging of intracellular ice crystallization events and stochastic analysis of the ice formation kinetics in a cell
population. Recommendations are provided for temperature profile design, sample preparation, and
configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate
best practices that have been drawn from two decades of experience with high-speed video cryomicroscopy
in our laboratory.
Key words Cryomicroscope, High-speed imaging, Ultra-slow motion, Intracellular ice formation,
Flashing, Nucleation, Kinetics, Cumulative probability, Cumulative hazard, Nelson-Aalen estimator
1 Introduction
The formation of ice within cells during freezing has long been
recognized as a major mode of cryoinjury [1]. In fact, a review of
experimental literature reveals a near one-to-one correspondence
between freezing processes that lead to irreversible cell damage and
those that result in intracellular ice formation [2], a correlation that
holds across a diverse range of cell types and freezing conditions: for
example, the critical cooling rate (the rate of cooling which, if
exceeded, causes the risk of intracellular ice injury to increase
above 50%) can differ by three orders of magnitude when comparing the freezing response of different cell species [2], whereas the
characteristic temperature of intracellular ice formation can range
from above À5
C to below À40
C, depending on cell type
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
221
High-Speed Video Cryomicroscopy for Measurement
of Intracellular Ice Formation Kinetics
Jens O. M. Karlsson
Abstract
Quantitative information about the kinetics and cumulative probability of intracellular ice formation is
necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues.
Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity
changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the
formation and growth of intracellular ice crystals can be accurately detected using temporally resolved
imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the
setup and operation of a high-speed video cryomicroscope system are described, including protocols for
imaging of intracellular ice crystallization events and stochastic analysis of the ice formation kinetics in a cell
population. Recommendations are provided for temperature profile design, sample preparation, and
configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate
best practices that have been drawn from two decades of experience with high-speed video cryomicroscopy
in our laboratory.
Key words Cryomicroscope, High-speed imaging, Ultra-slow motion, Intracellular ice formation,
Flashing, Nucleation, Kinetics, Cumulative probability, Cumulative hazard, Nelson-Aalen estimator
1 Introduction
The formation of ice within cells during freezing has long been
recognized as a major mode of cryoinjury [1]. In fact, a review of
experimental literature reveals a near one-to-one correspondence
between freezing processes that lead to irreversible cell damage and
those that result in intracellular ice formation [2], a correlation that
holds across a diverse range of cell types and freezing conditions: for
example, the critical cooling rate (the rate of cooling which, if
exceeded, causes the risk of intracellular ice injury to increase
above 50%) can differ by three orders of magnitude when comparing the freezing response of different cell species [2], whereas the
characteristic temperature of intracellular ice formation can range
from above À5
C to below À40
C, depending on cell type
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_8, © Springer Science+Business Media, LLC, part of Springer Nature 2021
221