Intracellular ice formation (IIF) plays a critical role in cryobiology, though the underlying biophysical mechanisms are still not completely explicable. In this work, a directional freezing scheme integrated with microlayer cell culture was employed to allow directional freezing as well as high-power microscopic observation of IIF in a single optical plane. The initiation of IIF and its spreading within the cells were well observed. The fluorescent reagents were employed to label the cell membrane and nucleus. It was found that the cell membrane could keep intact even though the cell had undergone IIF, and the intact cells could have IIF again in the next freezing cycle until their membranes were finally disrupted. The results shed light on the relationship between IIF and the integrity of cell membrane.

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