Slow-speed freezing of chemically unfixed biological tissues and long-term storage of frozen samples for cryoscanning electron microscopy.

We describe a procedure in which plant tissue as well as a yeast culture on agar are frozen with slow cooling rates for observation of surface structures in a cryoscanning electron microscope. A system is also presented for long-term storage of frozen specimens under liquid nitrogen, in which the material is maintained for direct observation. Some small tools are described, which are essential for making preparations using slow-speed freezing and for the storage of prepared samples. Three examples of preparations with different complications are given: the "sculptures" on the surface of a leaf of Allium schoenoprasum, an early stage of flower development of Allium cernuum, and a part of an agar-grown colony of Arxula adeninivorans. In our experience, it is possible to store fully hydrated samples under the described conditions for more than a year without damaging the fine structures.