Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation.

High-pressure freezing-freeze substitution (HPF-FS) methods were applied to germinating pea leaves. Good ultrastructural preservation without visible freezing damage was obtained up to 200 microns in thickness. Compared to conventional chemical fixation (CF), cellular membranes were smoother without undulation, and organelles appeared more turgid. The matrices of cytoplasm and organelles were denser and more homogeneous. The features imply that HPF-FS samples retain more substances and ultrastructure closer to the living state. There were differences in membrane stainability among organelles in HPF-FS specimens, which were not seen after CF. Bundles of microfilaments were observed frequently after HPF-FS.