Thermally defined cryomicroscopy and some applications on human leucocytes

A freezing-stage has been developed for use on a standard light-microscope, which can provide reproducible, precisely linear cooling and warming rates in the range from 0.1 to 10,000 K/min. Biological cells in aqueous solutions can be observed during the freeze-thaw cycle; the volume loss due to osmotic efflux of water and the intracellular crystallization of water are detected by video-monitoring. The temperature field generated in the observed samples is comparable to extended cylindrical probes and allows the transfer of cryomicroscopic data to technically used vial geometries. Lymphocytes and granulocytes were observed during freezing using the system described. They were separated and washed, and then frozen on the cold stage of the cryomicroscope at cooling rates ranging from 2 to 500 K/min. Shrinkage of the cells was observed up to 100 K/min and intracellular ice formation could be detected starting at 10 K/min. The results show that human leucocytes show excessive shrinkage up to 36% of their initial volume; the probability of intracellular ice formation exhibits a sharp increase from 10 to 100 K/min where nearly all cells contain ice.