Raman scattering evidence of hydrohalite formation on frozen yeast cells

TL;DR

This study uses Raman microspectroscopy to identify hydrohalite formation on frozen yeast cells, revealing how cooling rates influence eutectic crystallization patterns and potentially affecting cell injury during freezing.

Contribution

It is the first to link Raman spectral features to hydrohalite formation on cells and to analyze how cooling rates affect eutectic crystallization spatial distribution.

Findings

Hydrohalite identified as the source of the Raman peak near 3430 cm^-1.

Lower cooling rates produce thicker eutectic layers (~1 μm).

Higher cooling rates result in more homogeneous and thinner layers (~20 nm).

Abstract

We studied yeast cells in physiological solution during freezing by Raman microspectroscopy technique. The purpose was to find out the origin of a sharp peak near ~3430 cm^-1 in Raman spectrum of frozen mammalian cells, observed earlier (J. Dong et al, Biophys. J., 99 (2010) 2453), which presumably could be used as an indicator of intracellar ice appearance. We have shown that this line (actually doublet of 3408 and 3425 cm^-1) corresponds to Raman spectrum of hydrohalite (NaCl-2H2O), which is formed as the result of the eutectic crystallization of the liquid solution around the cells. We also show that the spatial distribution of hydrohalite in the sample significantly depends on the cooling rate. At lower cooling rate (1{\deg}C/min), products of eutectic crystallization form layer on the cell surface which thickness varies for different cells and can reach ~1 {\mu}m in thickness. At higher cooling rate (20{\deg}C/min), the hydrohalite distribution appears more homogeneous, in the sample, and the eutectic crystallization layer around the cells was estimated to be less than ~20 nm. These experimental results are consistent with scenarios predicted by the two-factor hypothesis for freezing induced cell injury. This work demonstrates a potential of Raman microspectroscopy to study peculiarities of the eutectic crystallization around single cells in vivo with the high spatial resolution.