运用激光扫描共聚焦显微技术研究大草履虫口胞器与消化胞器

Observation of oral organelle and digestive organelle in Paramecium caudatum using laser scanning confocal microscopy

本文利用激光扫描共聚焦显微技术(Laserscanningconfocalmicroscopy,LSCM)对大草履虫(Parameciumcaudatum)口胞器及消化胞器进行了再观察,通过连续断层扫描和三维重建技术,清晰地显示出草履虫口胞器中前庭、口腔、内口膜、四分膜、背咽膜、腹咽膜等结构,它们的位置和形态与前人的工作结果基本一致,并给出口胞器结构的三维立体构象。观察了虫体内的食物泡及其形成过程。看到胞咽在摄食并形成食物泡时其直径变大的现象,表明Allen(1974)有关消化后期食物泡产生的微小泡回到胞咽处与胞咽膜融合的时间应该是在胞咽直径扩大时,此时微小泡参与到胞咽膜上,进而再参与到新食物泡膜的形成过程中,由此完成膜的循环再利用。在样品制备中采用KMnO4替代了免疫荧光技术中传统的固定剂,固定效果很好,清楚地显示了胞咽膜。

Using laser scanning confocal microscopy, the morphological structures of oral organelles and digestive organelle in Paramecium caudatum were re-observed. In the preparation of samples, 5% KMnO4 was used. In addition, continuous tomoscan and 3-D reconstruction techniques were adopted. The result clearly revealed some structures of oral organdies such as vestibulum, buccal cavity, endoral membrane, quadrulus, dorsal and ventral peniculi, etc. Their positions and morphology basically corresponded with results in previous studies, 3-D conformation of oral structure is also provided. Food vacuoles and their forming process were also clearly observed. The diameter of the cytopharynx expands when it ingests and the food vacuole is formed. This suggests that the membrane of the vacuole may extend into the membrane of the cytopharynx just when the diameter of the cytopharynx expands. Specimens of P.caudatum were collected from a stream in the Wuchang region of Heilongjiang Province. The samples were prepared as follows： cells were suspended in 0.01% TritonX-100 solution for about 1 min, fixed by 5% KMnO4 solution for 4 min at room temperature, and washed in PBS （pH 7.4）. They were than exposed to 0.5% TritonX-100 solution for 25 min at room temperature; then incubated overnight in the primary antibody at 4℃ and washed in PBS. Avoiding sunlight, an overnight incubation in the secondary antibody at 4℃ follows, washed by PBS. Mounting the samples onto slides with glycerol [Acta Zoologica Sinica 51 （4） ： 718-722, 2005].