Researches On The Labeling Of GLUT4 Vecicles With Quantum Dots And The Real-time Positioning Of GLUT4 Vesicles In Live Cells With Nano-precision

Glut4 (Glucose Transporter 4), which plays a key role in insulin uptake in muscle and fat cells, is essential for maintaining whole body glucose homeostasis. The recycling of GLUT4 between the intracellular compartments and the cell surface involves many molecular mechanisms of endocytosis and exocytosis. A further investigation in GLUT4 translocation is helpful for revealing the basic life phenomenon of the across-membrane transport of macromolecules in live cells. However, only some short-term processes of GLUT4 translocation in partial aereas of the cell could be observed because of the limitations of the current 3D single particle tracking technique. This results that lots of important scientific problems on GLUT4 translocation can not be solved yet. By combining the new 3D single particle positioning technique with nano-precision with the quantum dots labeling technique, this study provides a platform suitable for long-term, real time mornitoring and analying on the GLUT4 trafficking in whole cell.The main works of this thesis are presented as follows:1. Quantum dots were introduced into the research of single particle tracking as an alternative to conventional dyes. A special protocol was developed to label GLUT4 in L6 cells and was demonstrated to be sensitive, specific, nontoxic. The results indicate that, compared with conventional dyes, quantum dots are advantageous in the research of 3D single particle positioning due to their unique optical and spectroscopic properties. Some difficult problems in 3D single particle positioning, such as low spacial resolution, low signal noise ratio, difficulties in object segmentation, short tracking time duration, have been solved by this method.2. Algorithms for 3D single particle positioning were investgated. Considering the request of real time and spatial resolution, we put the emphsis on investigating the relationship between the off-focus image and the off-focus distance. An approximately formula between the diameter of the outmost ring and the off-focus distance in Z-axis of single particle was established, which provided solid theory basis for real-time 3D single particle positioning with nano precision.3. A self-adjustable off-focus positioning system, including hardware and software platforms, was designed and established to perform real-time 3D single particle tracking. A piezocrystal mirco-displacement controller was designed to control the movement of object lens in Z-axis precisely. The Algorithm of real-time 3D single particle tracking was inserted to the software of TILLVision imaging system to meet the needs for real-time image acquisition and processing.The new method and experiment system of 3D single particle positioning established in this thesis can also be used in frontier science areas such as secretion mechanism of endocrine and neron cells, across-membrane transport of protein. With further development, this method has the potential of positioning and tracking protein macromolecules in live cells in real time and 3D.