Study Of Intracellular Transport And Exocytosis Of Vesicles Using Fluorescence Microscopy

The intracellular transport and exocytosis of vesicles are of critical importance for a variety of life functions and tightly related to some serious diseases. Deep study of the process of intracellular transport and exocytosis of vesicels will be helpful for understanding the mechinsms underlying cellular life functions as well as providing novel ideas and pathways for disease therapy. The emergence of new time-lapse fluorescence microscopies and the development of vesicle-specific stains have made the direct observation of the vesicle trafficking and exocytosis become of great interest among researchers around the world. Investigation of vesicle trafficking and exocytosis involves a lot of fields such as live-cell fluorescence microscopy, on-line successive imaging, patter reognition of digital image processing and computer simulation. This dissertation is mainly dedicated to using fluorescence microscopy to explore the three-dimensional mobility of single vesilce in live whole cells as well as the vesice exocytosis. It consists of following aspects: 1) Three-deimensional deconvolution microscopy is discussed. This mehtod uses wide-field fluorescence microscopy to collect three-dimensional images of particular specimens, and then employs the deconvolution algorithm to effectively reduce out-of-focus fluorescence in three-dimensional images. Deconvolution microscopy allows three-dimensional imaging with low photobleachig and photodamage, providing the solid basis for live-cell imaging. 2) By employing deconvolution microscopy, the spatial distribution of secretory vesicles in βcells and the secretory lysosomes in natrual killer cells are clearly obaserved. Experimental results demostrate that three-dimensional deconvolution microscopy is suitable for live-cell imaging. 3) Three-dinmensional single particle tracking is discussed. Centroid calculation-and gaussian-fit-based single particle tracking algorithms can follow the three-dimensional trajectories of single fluorescent particles with sub-pixel precision. Subsequently, the influence of optical imaging-associated diffraction limit on the performance of single particle tracking is quantitatively analysed. 4) By combining three-dimensional deconvolution fluorescence microscopy and single particle tracking, we succeed in following the three-dimensional mobility of single secretory vesicles in live whole PC12 cells. The population of vesicles is divided into two groups: the near-membrane group (within 1 μm from the plasma membrane) and cytosolic group. We evaluate and compare the three-dimensional mobility between these two groups under various physiological environments. This study surmounts the limit of previous methodologies in exploring single vesicle mobility and provides a novel approach to follow the intracellular trafficking of single vesicles in live cells. 5) Total internal reflection fluorescence microscopy can image live-cell images with relatively high temporal resolution, by which we directly monitor the mobility and exocytosis of near-membrane secretory vesicles and glucose transporter vesicles. Taking According to our study, it is expected that combination of deconvolution fluorescence microscopy and total internal reflection fluorescence microscopy will be helpful for exploring the intracellular trafficking of single vesicles throughout their entire life circle.