Automated image analysis for intracellular ice detection in monolayers by freeze-substitution

A major marker of cryoinjury is intracellular ice formation (IIF), and its prevention is a key design goal of a successful cryopreservation protocol. Visualization and automated image analysis of IIF will be helpful in gathering data for understanding the mechanisms of the freezing process. There is a need for high-quality frozen-cell images and a tool to quantify IIF for investigating the effects of various cryopreservation protocol parameters. Here, we report the development of a freeze-substitution-based method for light microscopy imaging of cryopreserved endothelial cell monolayers and an automated ice detection algorithm for high-throughput analysis of visible intracellular ice crystals in the nuclei of cells within the captured images. Our method detected ice crystals ≥1.10 ± 0.28 μm in the frozen-cell images and yielded comparable accuracy in ice detection to that achieved by manual counting. This study describes a tool for the identification and quantification of IIF visible under light microscopy.