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.
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Acknowledgments
The author gratefully acknowledges Peter Grocutt of Linkam Scientific Instruments for valuable discussions and technical support during the development of the high-speed video cryomicroscopy system and during the writing of this chapter. The original version of this work was supported by National Science Foundation grant CBET-1066619.
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Karlsson, J.O.M. (2021). High-Speed Video Cryomicroscopy for Measurement of Intracellular Ice Formation Kinetics. In: Wolkers, W.F., Oldenhof, H. (eds) Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, vol 2180. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0783-1_8
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DOI: https://doi.org/10.1007/978-1-0716-0783-1_8
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