【作者】 李栋栋；

【导师】 瞿安连；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2005， 博士

【摘要】 细胞分泌过程中囊泡的胞内转运和胞吐过程是细胞生命活动的基础,和许多疾病密切相关。深入研究囊泡胞内转运和胞吐过程不仅有助于了解细胞生命活动的机理,也有助于为疾病治疗提供新的思路和途径。随着荧光显微技术和活体细胞荧光标记技术的发展,直观观察和研究囊泡的胞内转运和胞吐过程已经成为国际上的一个研究热点。研究囊泡的胞内转运和胞吐涉及到活体细胞荧光显微成象、连续在线采样、数字图象模式识别方法和计算机仿真等诸多领域的问题。本文使用荧光显微技术研究了全细胞范围内囊泡的三维动态过程以及囊泡的胞吐,内容如下: 1) 讨论了三维反卷积荧光显微技术。该技术以宽场荧光显微镜和光学切片方法采集样本的三维图象,然后使用反卷积图象恢复算法有效地去除三维图象中的焦外光干扰。三维荧光反卷积显微技术能够实现低荧光漂白和低光毒性的三维成象,为长时程对活体细胞连续成象提供了坚实的基础。 2) 以三维反卷积荧光显微技术观察了胰腺β细胞内的分泌囊泡和NK细胞内的分泌性溶酶体的空间分布。实验结果说明三维反卷积荧光显微技术适用于观察活体细胞。 3) 讨论了三维单微粒跟踪技术。研究了基于质心计算和高斯拟合的三维单微粒跟踪技术,说明三维单微粒跟踪技术能够以亚象素级精度监测单个荧光微粒的三维位移。并定量分析了三维成象中的衍射限制对单微粒跟踪的影响。 4) 将三维反卷积荧光显微技术和单微粒跟踪技术有机地结合起来,成功跟踪了PC12细胞全细胞范围内单个分泌囊泡的三维动态过程。将细胞膜附近囊泡(相距细胞膜小于1 μm) 分为一组,细胞内其它囊泡分为另一组,深入研究并比较了不同生理条件下两组囊泡在动力学上的显著差异。该研究打破了以往显微成象和囊泡跟踪方法上的局限,为细胞内囊泡转运研究提供了新的技术手段。 5) 使用全内反射荧光显微技术观察了细胞膜附近分泌囊泡和葡萄糖转运蛋白囊泡的更多还原

【Abstract】 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.更多还原