TIME-VARIANT ANALYSIS OF ORGANELLE AND VESICLE MOVEMENT DURING PHAGOCYTOSIS IN PARAMECIUM-PRIMAURELIA BY MEANS OF FLUORESCENCE CONFOCAL LASER-SCANNING MICROSCOPY

Vital fluorescent dyes (FITC-albumin, Texas Red-albumin, and acridine orange) were used together with a confocal laser scanning optical micr oscope (CLSM) to display and analyze formation, movement, and fusion o f vesicles during the phagocytosis of Paramecium primaurelia, in the x -y-z-t space. By immobilizing living cells pulsed with a food vacuole marker at successive times after chasing in unlabeled medium, the intr acellular movement of food vacuoles from their formation at the cytost ome to their egestion at the cytoproct was visualized, and food vacuol es were selected in a specific digestion stage. Small pinocytic vesicl es are shown to evaginate from the vacuoles and move in the cytoplasm. These vesicles are transported toward the cytopharynx where they enla rge the membrane of the nascent food vacuoles or fuse with stage II fo od vacuoles, when the vacuoles of stage II increase their size, changi ng from an acidic to an alkaline status. A multimodal analysis of conf ocal fluorescence images and the false-color technique were used to vi sualize vesicle movement vs. time. Starting from three images of the s ame cell at succeeding time points, a composite image was generated by associating with each originally acquired image a different color cor responding to each sampling point in time. The composite image shows t hat vesicles move away from the food vacuole in a scattered manner exh ibiting changes in direction. (C) 1996 Wiley-Liss, Inc.