Analysis of Intracellular Freezing Process of Plant Cells with Cryomicroscope and High-Speed Video Camera

Normal-speed (32 frames/sec) and high-speed (up to 4,000 frames/sec) video cameras attached with a cryomicroscope were utilized to analyze the freezing process of protoplasts isolated from Arabidopsis leaves and Jerusalem artichoke tubers. With protoplasts isolated from leaves of non-acclimated Arabidopsis seedlings, intracellular freezing was observed even when protoplasts were frozen to -10℃ at rates of 0.5 to 1.0℃/min, and the frequency increased significantly at 2.5 to 10℃/min. After cold acclimation for 1 day at 2℃, the intracellular freezing did not occur at cooling rates of 1.0℃ or slower. Cold acclimation for 2 to 7 days further resulted in a shift of the temperature range over which intracellular freezing occurred to lower temperatures. High-speed video microscopy revealed that intracellular freezing was initiated, with no exceptions, at places near the plasma membrane. However, it was not possible with techniques used in the present study to determine whether ice formation initially occurs inside or outside the cell. Nevertheless, these studies demonstrate that videomicroscopy combined with cryostage provides an insight to understand the effect of cold acclimation on the freezing process of plant protoplasts