For compatibility with the electron microscope vacuum, and to lock the individual particles in place, the solution containing the sample material must be frozen. In order to preserve the macromolecular structures undamaged, freezing has to happen rapidly enough to avoid crystalline ice to occur, by rapid plunging. Then the sample has to be kept at liquid nitrogen temperatures at all times to preserve the amorphous nature of the embedding ice layer, and avoid damage to the biological particles.
This operation produces a frozen hydrated sample, where the individual molecules of the specimen are embedded in a very thin layer of amorphous (vitreous) ice and are well distributed within the ice.
The whole procedure can be simplified using semi-automated plungers such as the Thermo Scientific Vitrobot™. Based on a set of key parameters, such as specimen blotting time, blotting force, relative humidity, and temperature, this allows for reproducible preparation of vitrified specimens at high quality.