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.