High resolution view of the true cytosolic membrane surface of phagosomes of known ages purified from Paramecium

Techniques were used for viewing the true cytosolic surfaces of the membranes of intracellular organelles by field emission scanning electron microscopy (SEM). Cells of Paramecium multimicronucleatum were fed briefly with magnetic beads followed by a chase which advanced the newly formed digestive vacuoles (DVs) to predetermined ages. These bead-containing phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically; these DVs were similar to those in situ. The DVs prepared for transmission electron microscopy or SEM showed extensive adherence of cellular debris. The use of 0.2 M KCI in the wash buffer eliminated much of this debris and exposed the true vacuolar surfaces, Three populations of tightly bound vesicles and numerous globular particles of 10 to 20 nm became visible on the DV surfaces. The attached vesicles, hating diameters of similar to 300 and similar to 200 nm each, corresponded to the acidosomes and lysosomes that are known to be associated with the DV-I and DV-II, respectively. High resolution SEM also revealed a third set of small vesicles (50-150 nm), which were previously not known to be associated with DVs. The 10 to 20 nm globular particles were judged to be the cytosolic extensions of transmembrane protein complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these membranes in freeze-fracture studies.