Location: Biosciences Room 176

Freeze-substitution is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration. During freeze substitution the "frozen" water is dissolved by an organic solvent, which usually also contains chemical fixatives [1]. Freeze-substitution links instant physical immobilization of the cell constituents (cryo-fixation) and resin embedding. Once substitution is complete, samples are gradually warmed-up and processed further as for conventionally prepared samples. Successful cryo-fixation followed by FS shows superior preservation of fine structure compared to chemical fixation techniques [2]. Aggregation of macromolecules in organic solvents and changes of the hydration shell surrounding the biological molecules can occur even at very low temperatures, but it is reasonable to assume that FS at temperatures below a specific threshold preserves the hydration shell [3, 4].

This technique also gives the possibility of examining thick (200–300 nm sections) samples by ET, so that relatively large cellular volumes can be studied in 3D. This approach is very beneficial for an understanding of the complex relation between different cellular organelles and randomly occurring events.