Improved preservation of Amoeba proteus ultrastructure revealed by quick-freezing followed by freeze-substitution

Abstract

Summary For better preservation of Amoeba proteus ultrastructure, we have applied the quick-freezing and freeze-substitution method to them which are difficult to fix optimally for conventional electron microscopy. This method provided a greatly improved visualization of A. proteus when compared with the conventional fixation: (1) Most of the membrane components including the cell membrane and intracellular membranes were smoother and showed distinct trilaminar substructures. Only the vacuolar membrane containing a crystal had wrinkles; (2) substructure of the surface coat of the cell membrane was clearly distinguishable; (3) cytoskeletal components were well preserved in the cortical and central cytoplasm; (4) each Golgi component and cistern of the endoplasmic reticulum (ER) were more clearly resolved, and their contents were also well preserved; (5) variously sized Golgi vesicles associated with the trans face of the Golgi network (TGN) were clearly preserved. Only clathrin-coated vesicles were derived from the TGN; (6) perivacuolar vesicles with contractile vacuoles and satellite vesicles around the food vacuoles were clarified, and all of their contents were well preserved; (7) mitochondrial, cytoplasmic, and nuclear matrices were denser and filled with an abundance of fibrillar and granular materials. Consequently, after comparing the above findings with information obtained by the method using conventional chemical fixation, we suggest that all theses observations obtained by the quick-frozen / freeze-substitution method indicate a more faithful representation of A proteus ultrastructure.