Ultrastructure of early chick embryo tissues after high pressure freezing and freeze substitution

Early chick embryos (stages 11-29), or parts thereof, were frozen by the high pressure technique and subjected to a variety of freeze substitution protocols. In addition to hexadecene, unincubated albumen was used as a cryoprotectant to surround the specimens during the freezing event. The quality of freeze ranged from poor to very good in these highly aqueous specimens; very good freeze quality was observed minimally to depths of 30-40 microns. Freeze substitution was carried out with a custom-made aluminum chamber, automatic freeze substitution machine, or a combination of the two, in acetone containing 1) uranyl acetate; 2) acrolein and/or tannic acid; or 3) a sequence of 2), osmium, and glutaraldehyde. Excellent quality of morphology was cryopreserved with all solutions, given optimally cryoimmobilized tissue. Mitotic and pericentriolar microtubules and cytoskeletal elements were cryopreserved with all methods; membranous organelles were better preserved with osmium. Outstanding preservation of morphology of the ground substance of very hydrated embryonic cells, though limited in depth, was accomplished by high pressure cryoimmobilization and cryopreservation, demonstrating that the technique can be successfully applied to highly aqueous tissues.