Intracellular ice formation (IIF) is of great importance in cryobiology but its mechanisms are still under debate. In this work a micro-layer chip with a sandwiched thin layer of medium was employed to facilitate the observation of IIF; and thermoelectric chips were used to induce a directional cooling along the chip. Remarkable phenomena of extracellular ice formation (EIF) triggering IIF were observed. The extracellular ice contacted and "penetrated through" and initiated the intracellular ice. In the aid of the fluorescent reagents, it was found that the cell membrane could keep intact even though the cell had undergone IIF, and the intact cell could have IIF again in the next freezing cycle, until the membrane was finally ruptured. The results shed light on the IIF mechanisms.
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