Spatial Distribution of the State of Water in Frozen Mammalian Cells

We describe direct determination of the state of intracellular water, measurement of the intercellular concentration of a cryoprotectant agent (dimethylsulfoxide), and the distribution of organic material in frozen mammalian cells. Confocal Raman microspectroscopy was utilized at cryogenic temperatures with single live cells to conduct high spatial resolution measurements (350 × 350 × 700 nm), which yielded two, we believe, novel observations: 1), intracellular ice formation during fast cooling (50°C/min) causes more pronounced intracellular dehydration than slow cooling (1°C/min); and 2), intracellular dimethylsulfoxide concentration is lower (by as much as 50%) during fast cooling, decreasing the propensity for intracellular vitrification. These observations have a very significant impact for developing successful biopreservation protocols for cells used for therapeutic purposes and for cellular biofluids.