Fok A K, Ma L, Aihara M S, Allen R D
Biology Program, University of Hawaii, Honolulu 96822, USA.
Eur J Cell Biol. 1996 Nov;71(3):259-69.
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 KCl 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, having diameters of approximately 300 and approximately 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.
利用场发射扫描电子显微镜（SEM）技术观察细胞内细胞器膜的真实胞质表面。用磁珠短暂喂食多核草履虫细胞，随后进行追踪，使新形成的消化液泡（DVs）发育到预定年龄。用磁铁从全细胞匀浆中分离出这些含珠子的吞噬体，并通过荧光显微镜确定其完整无损。从抗原性来看，这些DVs与原位的相似。为透射电子显微镜或SEM制备的DVs显示出大量细胞碎片附着。在洗涤缓冲液中使用0.2 M KCl可去除大部分此类碎片，暴露出液泡的真实表面。在DV表面可见三类紧密结合的囊泡以及众多直径为10至20 nm的球状颗粒。附着的囊泡直径分别约为300 nm和约200 nm，分别对应于已知与DV-I和DV-II相关的酸小体和溶酶体。高分辨率SEM还揭示了第三组小囊泡（50 - 150 nm），此前未知其与DVs相关。直径为10至20 nm的球状颗粒被判定为跨膜蛋白复合物的胞质延伸，因为它们在不同年龄DVs上的分布模式与之前在这些膜的冷冻断裂研究中看到的跨膜颗粒相对应。
