The recorded image data of our study consist of a complete Raman spectrum per pixel. From these data chemical maps of the contained compounds can be extracted. Subsequently, color coded overlay images can be prepared and utilized to determine the spatial distribution of hydrohalite and cellular matter. In some cases the overlay images are ambiguous with respect to the hydrohalite localization – mostly due to the limited axial resolution – and specific characteristics in colocalization plots are found to be helpful in the further interpretation of the data. Spatial correlation between hydrohalite and cellular matter

will show up in colocalization plots and can be used to determine whether the hydrohalite is located within or outside the cell. It is indeed this website shown, that hydrohalite can form inside cells under certain conditions, though it seems less serious in established cryopreservation protocols in vital biobanking. However, it has to be considered in the study of cryoinjury mechanisms. The

experimental setup consists of three elements; A confocal Raman microscope, a temperature controlled chamber Apoptosis inhibitor and a scanning stage. We measured the point spread function giving a radial and axial FWHM of 0.8 μm and 2.5 μm for the optical setup. Further details on the experimental setup can be found in [10]. For the example Raman spectra of Me2SO and cellular matter shown in Fig. 1a two samples at room temperature containing either pure Me2SO (WAK-Chemie GmbH, Germany) or mouse fibroblasts in PBS (PAN Biotech GmbH, Germany) were used. Two additional samples were used for the Raman spectra of ice and hydrohalite, which was recorded at a temperature of approximately −20 °C using solutions of 25 wt.% NaCl saline solution or demineralised water. The integration time for these Raman spectra is 2 s. The Raman images are recorded using adherent mouse fibroblasts in

PBS (PAN Biotech GmbH, Germany) and are cooled to −50 °C at a cooling rate of −1 °C/min. The integration time for each pixel is 100 ms and the Orotic acid images have a scan area of 50 μm × 50 μm. The investigated samples were equilibrated a few minutes in either PBS without Me2SO or with 0.5 wt.% Me2SO at room temperature before the cooling protocol were applied. The sample volume was approximately 10 μL, which corresponds to a sample height of ≈40 μm. The investigated cell line is the L929 mouse fibroblast from ATCC (United States). The cells were incubated at 37 °C and a 5% CO2 atmosphere in Gibco© Dulbecco’s modified Eagle medium (Life Technologies, United States) with 10% fetal calf serum on glass cover slips (VWR, United States). The cells were handled using standard procedures. We use confocal Raman microscopy to investigate the solid states that form in cryopreservation samples upon cooling. The Raman spectra of the compounds encountered in this study are shown in Fig. 1a.