A method for rapid freeze fixation of plant cells

Summary We describe here an apparatus that permits rapid freeze fixation of whole cells, which are then prepared by freeze substitution and resin embedment for examination in the EM. The freezing device utilizes a rotary solenoid that rapidly plunges the specimen holder, a formvar-film-covered thin wire loop, into a well of stirred liquid propane at –180C. The rotary solenoid allows for an adjustable, repeatable immersion rate. Substitution takes place at –80 C in acetone with 2% OsO₄ and is followed by en bloc staining in either hafnium tetrachloride or uranyl acetate. We have utilized these techniques on plant cells, for which there has been relatively little published work when compared to other organisms. The results show that, with the versatile specimen holder and rapid, repeatable immersion rates, different cell types, including pollen, stamen hairs, and germinating moss spores, can be rapidly frozen with repeatable success. The improved preservation achieved with rapid freeze fixation over conventional chemical fixation reveals itself particularly in the structure of the plasmamembrane, the cytoskeleton, chromatin, and certain endomembrane systems.