An optical-axis freezing stage for laser-scanning microscopy of broad ice-water interfaces.

This article presents a method to view a dynamic ice interface along the axis of ice growth using a laser-scanning microscope. A deep liquid volume is chilled from below so that ice growth is directed upward toward the microscope objective. The interface is made visible by rejection of fluorescent dye from the solid phase into the liquid. Images of the interface morphology in water with solutes of interest to cryobiology illustrate the imaging capability. These images are processed to quantify the lamellar structure of the ice interface. The optical-axis cryostage provides advantages over horizontal arrangements because (1) immersion objectives enhance, rather than disturb, the desired thermal gradient, and (2) features in the ice interface are not confined within a narrow capillary tube or microscope slide. This arrangement loses some of the thermal control found in planar freezing stages, and the dynamic, refractive interface presents challenges to confocal microscopy.

Measurement of cell volume loss in the liquid region preceding an advancing phase change interface.

It is well understood that the solidification of a solution results in a redistribution of solute in the liquid zone. For the freezing of suspensions of cells it is anticipated that accumulation of solute in the region leading a growing ice phase will cause an osmotic response in cells before the ice phase reaches the cells. To measure this phenomenon in a specific algal species, the volume changes in Chlorococcum texanum during freezing were studied using directional solidification cryomicroscopy. The relative cell volume was tracked continuously as a function of temperature and position as cells encountered the moving phase front. The loss of cell volume was measured in the liquid region containing concentrated solute ahead of the growing solid phase.