Pre- and post-thaw assessment of intracellular ice formation.

Intracellular ice formation (IIF) refers to the formation of ice crystals within cells during rapid freezing. To develop an understanding of the means by which intracellular ice forms and the mechanisms by which it damages cells and tissues requires techniques that combine real-time assessment of ice nucleation and ice crystal growth with detailed assessments of cell structure and function. Intracellular ice formation has been detected in live samples using light scattering, freeze substitution and fluorescent detection. In this study we develop a method to correlate IIF with post-thaw structural analyses by combining low temperature microscopy and freeze substitution. V79-4 hamster fibroblasts were frozen on a low temperature microscope at various temperatures, IIF was visualized using the nucleic acid-specific fluorophore SYTO 13, then the samples were fixed (10% formaldehyde, 85% ethanol, 5% acetic acid) while still frozen. The monolayers were then thawed and stained with routine histological stains haematoxylin and eosin and assessed. Fixation allowed for the post-thaw assessment of IIF and for subsequent histological processing to examine in detail the structural consequences of IIF. The post-thaw identification of cells that form intracellular ice during freezing is a significant improvement to current methods used in low temperature biology.