  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.
