Research On Technique For Real-time 3D Localization Of GLUT4 Vesicles In Live Cells With Nano-precision

The Glucose transporter 4 (GLUT4) plays a key role in glucose uptake in skeletal muscle cells and adipocytes. GLUT4 can translocated to the cell membrane upon insulin stimulation, and promote the uptake of glucose. Imbalance of this process can lead to typeâ…¡diabetes. Observing the translocation of GLUT4 in live cells is important for study the pathogenesis of typeâ…¡diabetes. Because of the limitaitions of single particle 3D localization technology and the defect of organic dyes and fluorescent proteins, relative weak fluorescent signal against strong cellular autofluorescence background and fast photobleaching rate, most studies only focused on particular segments of GLUT4 traffic. Long-term localization of GLUT4 in 3D space and time can provide new insights for understanding the mechanisms of insulin-regulated GLUT4 translocation. In this paper, we dedicate to research and develop a technique for localization of GLUT4 in live cells in 3D space and time, to provide a new method for observing GLUT4 translocation in live cells.In this paper, GLUT4 is labeled by quantum dots (QDs) in live cells. Due to the advantages of QDs, such as broad absorption spectrum, a narrow emission spectrum, very high levels of brightness and photostability, GLUT4-QDs overcome the limitations that conventional dyes suffer from. Furthermore, real-time single particle 3D localization system can be constructed by using the relationship of off-focus image and defocus distance. The main parts of this paper are as below:(1) Experimental method for labeling GLUT4 in live cells using QDs. GLUT4 on live cell membrane can be labeled by QDs by adjusting reaction temperature and concentration of QDs, and the translocation of GLUT4 from cell membrane to the intracellular organelle can be observed. The advantage of this method, such as specificity, high sensitivity, low toxicity and easy to be detected, can be proved by experiments.(2) Algorithm of real-time single particle 3D localization. The relationship between the radius of outmost ring in off-focus image and defocus distance can be calculated. Furthermore, the image processing algorithm of real-time single particle 3D localization in nanometer precision, containing pretreatment, iterative centroid algorithm and calculation of outmost ring in off-focus image, is designed.(3) Research and develop of the real-time single particle 3D localization system. To put the algorithm of real-time single particle 3D localization in use, a real-time single particle 3D localization system is designed. This system contains tow parts, hardware part and software part. The hardware part is consist of instruments which designed by our lab, inverted microscope and CCD. The software part is consist of control software for all parts of hardware and the algorithm of real-time single particle 3D localization.