High‐pressure freezing of plant cells cultured in cellulose microcapillaries

A new microculturing technique for plant cells was used to meet the requirements of high‐pressure freezing (HPF). The plant cells were cultured inside cellulose microcapillaries, providing an easy‐to‐handle method for a real in situ fixation. The high viability of the cells was demonstrated by regenerating shoots from microcalluses cultivated by this method. In general, the freezing quality of the high‐pressure frozen samples was excellent across the whole diameter of the capillaries, as shown with ultrathin sectioned cells after freeze‐substitution and embedding in Spurr's resin. In comparison with conventional chemically fixed cells, cultured under identical conditions, all membranous compartments and organelles were more turgid and smoother after HPF. The cytoplasm and the matrix of the organelles were more homogeneous and dense. Thus, high‐pressure freezing in combination with the microculture method described here appears to preserve the ultrastructure of chemically untreated plant cells close to the native state.