C-10: High-speed video imaging of ice crystal formation in cells, tissues and droplets

Attempts to elucidate the causes of cryoinjury have benefited from nearly two centuries of microscopic observation of cell and tissue response during exposure to cold temperatures. However, the mechanisms of intracellular ice formation, a phenomenon that is strongly associated with irreversible cell damage, remains poorly understood: even in suspended, isolated cells, it has not been possible to obtain experimental evidence that conclusively supports or contradicts any of the competing hypotheses of intracellular ice formation (viz., the surface-catalyzed nucleation model, pore theory, or membrane rupture hypothesis). In part, this lack of progress has been due to the limited temporal resolution of conventional cryomicroscopy technology. Until recently, image acquisition rates in video cryomicroscopy studies have typically not exceeded 30 frames per second (fps), yielding a temporal resolution of 33 ms. Because the transformation of cell water to ice is completed in approximately 1 ms or less during rapid-cooling experiments, intracellular ice formation events cannot usually be detected using conventional video-micrography. As a result, cryomicroscopists have relied on the observation of cytoplasmic opacity changes as a proxy for intracellular freezing, an approach that can cause significant errors in estimates of intracellular ice formation kinetics. To detect the initial appearance and growth of ice crystals inside supercooled cells, and to quantify the phase transformation kinetics, it is necessary to use high-speed video cryomicroscopy to acquire micrographs at sub-millisecond resolution. In particular, to accurately measure intracellular ice formation kinetics in rapidly cooled somatic cells, image acquisition rates in the range 10 3 –10 4  fps are required, whereas shutter speeds should be faster than ∼0.1 ms to prevent motion blur. We have implemented such a high-speed imaging cryomicroscopy system, and have successfully used it to visualize non-equilibrium ice crystal formation and growth inside cells, tissue constructs, and microscale water droplets.