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

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