5 wt% Me2SO has been added to the cell medium. This dramatically changes the equilibrium phase diagram since Me2SO also will be concentrated in the unfrozen interdendritic channels [9]. Hydrohalite was only observed in two samples out of six, where one only contained a very limited amount of hydrohalite, which is in stark contrast

to the experiments not using Me2SO. The lack of hydrohalite is unexpected since the phase diagram and earlier studies show that hydrohalite can form in hypertonic solutions with a higher Me2SO to NaCl ratio as a continuous precipitation process [10]. This study is done on an isotonic solution, which in equilibrium would form hydrohalite at these temperatures, but has much narrower interdendritic channels compared to a hypertonic 5-Fluoracil chemical structure solution. Two kinetic factors can limit the formation of hydrohalite; viscosity and impeded diffusion due to narrow interdendritic channels. The viscosity in the unfrozen solution is high due the presence of Me2SO and the low temperatures. Diffusion afflux to any hydrohalite crystal embryos is furthermore limited due to the very low interdendritic cross sections. We believe that

a combination of these two factors prevented hydrohalite formation in the majority of the investigated samples. Three of the recorded Raman images for the one sample containing a significant amount of hydrohalite are shown in Fig. 5. The recorded images can be divided Alectinib cost into classes using the categorization method presented earlier. Fig. 5a show cells

where there is no overlap between cellular matter Quinapyramine and the hydrohalite phase, i.e. Class A. In total 3 out of 6 images contained clearly extracellular hydrohalite. Fig. 5b and c does on the other hand show a certain spatial overlap of compound distributions, but not in a significant manner that we would correlate to intracellular hydrohalite. The distribution of hydrohalite in these Raman images can be best classified to Class C for Fig. 5c and a superposition of Class A and C for Fig. 5b using the colocalization method. We have shown that confocal Raman microscopy can be utilized to extract detailed chemical information of frozen biological samples. In samples without Me2SO we used this method to determine the distribution of hydrohalite and thus indirectly conclude if eutectic formation has occurred. It turns out that hydrohalite can either form in the very close proximity of cells as non-uniform shell or even intracellularly. Hydrohalite is thus not a strictly extracellular phenomenon. Furthermore, we showed that hydrohalite has a higher probability of forming within the cytoplasm when ice is also present. Eutectic formation in general has been shown to lead to cell death [8], but the exact injury mechanism has not been determined. We have shown that hydrohalite formation, and thus eutectic formation, can occur both within and outside cells, which can bring a more detailed view on the mortality of eutectic formation.