The main purpose of the proposed research is to construct, test and evaluate a prototype of high pressure-ultrafast freezing apparatus similar to the one developed by Professor Hans Moor in Zurich, Switzerland. Theoretically, this apparatus, which can pressurize samples to 2,000 atmospheres in 20 msec before freezing, should be able to virtrify samples of up to 0.5 mm in diameter, but this needs to be confirmed by an independent laboratory using variety of biological samples before commercial development of the instrument can occur. We plan to perform such evaluative research as well as to explore possibilities for improving the processing of ultrarapidly frozen specimens for freeze-fracture (-etch), for freeze-substitution, for freeze-drying, for cryosectioning and for cytochemical studies. Much of the impetus for developing improved specimen preparation techniques will come from studies on transient or labile membrane and cytoskeletal structures in animals and plants, where the application of ultrarapid freezing techniques could lead to significant advances in our understanding of cell function. The four biological topics to be investigated are: a) Structural and physiological responses of a newly discovered membranous organelle believed to be involved in osmoregulation in Pelvatia embryos. b) Membrane dynamics of endoplasmic reticulum-Golgi-GERL-lysosome systems in selected animal, plant and algal cells and tissues. c) Dynamics of the mitotic apparatus and of associated membrane systems in Drosophila eggs. d) Microtubule-regulated directionality of cell wall fibril deposition in plants and algae.
