Hydrohalite spatial distribution in frozen cell cultures measured using confocal Raman microscopy.

Hydrohalite, a crystalline rock salt hydrate, (NaCl·2H2O), can form in cryopreservation samples under certain circumstances changing the local chemical environment of the preserved cells. Evidence of this crystalline phase was recently found by microspectroscopy measurements, and believed to form exclusively extracellular. We have studied the spatial distribution of hydrohalite in frozen mouse fibroblast cell samples by means of confocal Raman scanning microscopy (CRM). Hydrohalite has a unique Raman spectrum with several bands in the high frequency tail of the OH-stretching band which can be used for unambiguous identification. Hydrohalite can only form through eutectic crystallization in saline solutions without any cryoprotective agents and the spatial distribution thus gives a more detailed view on this crystallization process. This is important since eutectic crystallization has been empirically correlated to cell death, but the exact injury mechanism is unclear. By the means of colocalization of Raman bands we show that hydrohalite can indeed form intracellularly and is not a strictly extracellular phenomenon. We furthermore found that intracellular ice and intracellular hydrohalite very often coincide. Finally we show that the addition of 0.5 wt.% dimethyl sulfoxide (Me2SO) inhibits formation of hydrohalite. This study shows how Raman microscopy and successive analysis can be employed non-invasively within cryobiology to give additional chemical and structural information compared to conventional imaging techniques.