Two methods of the in-situ observations of ice crystal growth from the supercooled water including impurity are briefly summarized. The free growth method of crystal is very useful to observe the growth as a function of the driving force for crystal growth (namely, supercooling temperature). The one-directional growth method is often used to observe the morphological instability and the pattern formation. As a typical example of the in-situ observation, the pattern formation at the interface between ice and KC1-solution is analyzed by the way of Mach-Zehnder interferometer. The mechanism of the morphological instability is discussed as the effect of impurity included in the water.

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Micro-behavior of ice crystals and cells in the cell suspensions and the tissues during the freezing and thawing was visualized in three-dimensions using a confocal laser scanning microscope (CLSM) and a fluorescent dye. The CLSM noninvasively produced tomograms of the biological material on real time. Freezing pattern, morphology of ice crystals, and ice-cell interaction in the following three different processes were investigated qualitatively and analytically from the mechanical point of view: 1) directional solidification (extracellular-freezing) of human red blood cell suspensions, 2) directional-solidifications parallel and perpendicular to the direction of muscle fibers of the fresh pectoral muscles of chicken, and 3) recrystallization of ice crystals during the slow-warming of the rapidly-frozen muscle tissues.

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The process of cold acclimation in plants, which elicits an increase in freezing tolerance, is a complex developmental phenomenon that involves the orchestration of many different processes. Although seemingly desperate, many of the cold acclimation-associated processes ultimately contribute to the increase in cryostability of the plasma membrane, destabilization of which is the primary cause of freezing injury. To understand cold acclimation process comprehensively, we need to characterize the freeze-induced lesions that determine the freezing tolerance and then the mechanism of minimization of these freeze-induced lesions during cold acclimation. In this review, we describe the specific freeze-induced lesions in isolated protoplasts and then several important changes occurring during cold acclimation, which are associated with the minimization of the freeze-induced lesions and, hence, the increase in freezing tolerance. This approach allows us to make strategic plans for molecular breeding of cold-tolerant agricultural plants efficiently.

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High resolution nuclear magnetic resonance imaging (i.e., NMR microscopy) was used to study the freezing behaviors in wintering flower buds of azalea (Rhododendron japonicum), leaf and flower buds of full-moon maple (Acer japonicum) and leaf buds of Momi fir (Abies firma) and lilac (Syringa vulgaris). The images acquired predominantly reflected the density of mobile (i.e., non-ice) protons mainly from unfrozen water. By comparing these images taken at various subfreezing temperatures, we could successfully visualize typical extra-organ freezing in the florets of R. japonicum and shoot primordia of maple and fir and deep supercooling in the maple xylem and azalea pith. Extracellular freezing was also clearly seen in the bud scales and bark tissues of these species and primordial leaves of lilac. To correctly interpret the images, all the factors that affect image intensity at differing temperatures are considered but the water to ice transition is the most dominant. Other problems that may be encountered in the interpretation of the images are also considered. The results of our studies show that NMR microscopy allows dynamic and species specific freezing processes of various plant tissues in a single specimen to be non-invasively visualized. Further, the information obtained provides deeper insight into the diversity and mechanisms involved in freezing behaviors.

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Xylem ray parenchyma cells in woody plants adapt to freezing of apoplast water by deep supercooling. The mechanism of deep supercooling is proved not due to merely cell wall property that water in micro-capillaries within the cell walls are sufficiently small to keep supercooling by which inhibits ice seeding of protoplasts through the cell walls. The mechanism of supercooling, thus, may respond to intracellular factors, such as accumulation of any substances that facilitate supercooling. Our approach to identify these intracellular substances from xylem ray parenchyma cells of woody plants that exhibit deep supercooling was introduced in this review.

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