Conventional chemical fixation methods require the removal of water, the main constituent of most biological samples, and often result in various artifacts. Solidification of aqueous samples by lowering their kinetic energy, i.e., freezing, is an alternative physical means for immobilizing the samples. However, if water in the sample crystalizes into ice upon freezing, the structure of the sample would be destroyed. Thus, in order to preserve the native structure of the sample, freezing should result in vitrification, that is, the solidification of water in an amorphous state, rather than crystallization. When water is frozen under pressure of 2,100 bars, the crystallization process is slowed down and freezing may result in vitrification provided that the sample is thin enough (< ~200 µm) in order to allow for cooling to be fast. The application of high pressure freezing (HPF), allows for the vitrification of biological as well as synthetic hydrated samples. During HPF, all constituents of the sample are literally frozen in space and time, thus the investigated structure of these samples closely reflects their native structure. Once vitrified by HPF, samples can be further processed for or visualized by techniques such as cryo-SEM, cryo-TEM, STEM tomography, freeze substitution etc.

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