This will lead to an inverted “U”-shape, which was observed along

This will lead to an inverted “U”-shape, which was observed along with extracellular hydrohalite shells as opposed to the linear correlation in case of intracellular hydrohalite formation. We recorded 24 confocal Raman images as the one shown in Fig. 1e distributed on four different samples containing L929 mouse fibroblast cells without Me2SO. All images except one contain hydrohalite found over the entire sample. The last image does not contain hydrohalite. We also investigated 6 samples with Me2SO, but only found a significant amount of hydrohalite in one, of which we recorded 6 Raman images. Each Raman image contained primarily one cell, but images with

up to three cells were

also recorded. All samples were subjected to identical freezing protocols. A typical transmission Akt inhibitor (TM) image and the corresponding Raman responses from cellular matter and hydrohalite are shown in Fig. 1b–d. These images contain one cell and an interdendritic channel. This can however not directly be concluded from the TM image alone. The Raman images reveal that the dendritic channel contains a high amount of hydrohalite and no cellular C646 concentration matter, whereas the hydrohalite phase overlaps the Raman response from the cellular matter. It can furthermore be concluded from the Raman images that the investigated cell contains a large aminophylline intracellular ice crystal, since most of the cellular matter is displaced towards the rim of the cell, and this displacement can only be attributed to intracellular ice crystals. These features cannot readily be seen from the TM image and clearly demonstrates how Raman imaging gives both more structural and chemical information compared to conventional imaging techniques. We found that the recorded Raman images of the samples without Me2SO can be roughly divided into three different classes, exemplified by the Raman images in Fig.

3a–c. Overlaps between the groups do however exist and some images are attributed to multiple classes. The first class, denoted Class A, contains images with very little or no overlap between the hydrohalite phase and cellular matter. This can readily be seen in the Raman images as in Fig. 3a. The hydrohalite in these images are thus clearly extracellular, although in close proximity to the cell. We found that 6 images out of the 24 contained extracellular hydrohalite. The two remaining classes, denoted Class B and Class C, contain Raman images with overlapping hydrohalite phase and cellular matter, i.e. data points where the focal volume contains both hydrohalite and cellular matter. Class B is defined to contain intracellular hydrohalite whereas the hydrohalite is located outside the cell for Class C. Two more examples of recorded Raman images are shown in Fig.

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