Abstract
Interactions among water, ice, and solute in a frozen organic solution play an important role in stability of the organic matter during cryopreservation. The aim of this study was therefore to investigate microscopic distribution of water, ice, and organic matters after freezing. A lysozyme aqueous solution was used as a sample with trehalose as a cryoprotective agent. The binary solution or each component solution was frozen in a microchannel on a directional solidification stage combined with a laser Raman microscope, and observed to obtain spatial distribution of C-H bond, O-H bond and Amide I band. Lysozyme in its solution was finely dispersed at highly subcooled areas, while formed condensed spots at the lower subcooled areas as a result of being rejected from ice crystals. Trehalose solution however showed the condensed spots even at the highly subcooled area. The binary solution also showed the distribution of solutes similar to the trehalose solution. The important feature was colocalization of trehalose and lysozyme, which might be favorable for preservation of lysozyme due to the cryoprotective effect of trehalose.
