Laser scanning microscopy of broad freezing interfaces with applications to biological cells

A new, vertical cryostage was used for microscopic observation of broad-front freezing in aqueous solutions. This cryostage complements traditional studies of cell behavior and interface morphology in cryobiology. Traditional systems directionally solidify thin samples perpendicular to the optical axis. Thin samples confer thermal and optical advantages for video brightfield microscopy. However, sample thickness can affect the interface morphology.;In the new cryostage, ice propagates parallel to the microscope optical axis. The sample cup is 1 cm tall and 1.5 cm in diameter, with insulated sides and a nitrogen-cooled base to freeze the solution upward. The top of the solution is warmed passively through a cover glass or immersion objective. The freezing solutions contain dilute fluorescein dye, which is visible where it is concentrated by exclusion from the ice. The stage is mounted on a confocal laser-scanning microscope, and thermal control and image capture routines are centralized in a LabView user interface.;Filtered water, physiological saline, 9.5% glycerol, and 10% glycerol with PBS were frozen at rates between -2°C/min and -10°C/min and sequential images at one plane were captured. Images distinctly revealed a lamellar interface but could not resolve 3-D morphology. The average lamellar spacing was quantified using image analysis.;Physiological saline was frozen in flat glass capillary tubes with 0.05 to 0.4 mm path length, mounted vertically to observe internal ice in cross-section. Lamellae were randomly oriented with respect to the glass, suggesting caution when measuring dendrite spacing in a horizontal cryostage. No correlation between capillary size and lamellar spacing was noted.;Cell monolayers and synthetic membranes were mounted horizontally to let a well-developed ice front approach the layer broadly. In transparent membranes, ice-membrane interaction was visible until ice grew over and obscured the membrane.;The vertical cryostage improved our ability to observe ice lamellae in cross section. The broad phase front revealed dendrites' interaction with each other, rather than with the container walls. The arrangement permitted immersion objectives, which are normally avoided in cryomicroscopy because of heat transfer. The signal-to-noise ratio was too low for effective confocal microscopy, but laser-scanning microscopy provided good contrast compared to standard epi-fluorescence methods.