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<p>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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月9日

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  • Ming Han Yu,
  • Leah A. Marquez-Curtis,
  • Janet A. W. Elliott

摘要

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.