TEM microscopy works under vacuum and requires essential steps of sample preparation. The first step is the fixation of the biological sample to preserve its structure and to "freeze" physiological processes in a snapshot. There are different fixation methods, such as denaturation by formaldehyde, cross-linking by glutaraldehyde or displacement of water and denaturation by alcohols. Secondary fixation with osmium tetroxide enhances the contrast of lipid membranes. Vitrification of a thin sample at less than - 150°C preserves the ultrastructure almost artefact-free. However, cryo-fixation results in weaker contrast relative to chemical fixation with contrasting.