A gentle bounce-free assembly for quick-freezing tissues for electron microscopy: application to isolated torpedine ray electrocyte stacks.

Thin, isolated stacks of Narcine brasiliensis electric organ were prepared for freeze substitution using a rapid freezing device similar to that described by Van Harreveld and Crowell (1964). Although the impact stress was reduced as much as the design of the apparatus allowed, the tissue was found to become severely squashed and the first layer of nerve terminals was found to be badly damaged. However, in occasional blocks, the morphology of the deeper layers encouraged the view that the tissue could be well suited to the procedure if the impact squash could be minimized. A second problem was identified when studying the impact of various tissue holders with the freeze surface: the original type of apparatus is highly susceptible to bouce in the millisecond time range. Tissue squash was controlled by mounting the sample on a piece of dry foam fitted into a slotted striker tip. Bounce was eliminated by coupling the striker to a simple hydraulic-pneumatic damping device. When electrocytes were frozen with this apparatus and freeze-substituted, the first layer of nerve terminals was found to be intact, well frozen and well fixed.