| Summary: | 碩士 === 國立臺灣大學 === 醫學工程學研究所 === 99 === In this thesis, we use the two-photon based fluorescence correlation spectroscopy system to measure the dynamic parameter of green fluorescence protein in PBS, and in cytosol. The concentration of GFP was increased with time during the period of the time, 6-27 hours after transfection. According the correlation we should know our system limitation of the concentration GFP. Our purpose is to set up a platform which is low bio-damage and for long-term observation for the study of the cell biology such as cytoskeleton and transportation in cell which are related to the metastasis of cancer cell or cell movement. In general, the studies of cytotskeleton or membrane transfusion rate are use Fluorescence recovery after photobleaching (FRAP) as a tool to study. The laser of FRAP was one photon excitation. Compare to the one photon excitation two photon excitation had less photobleaching, less cell damage and longer penetration depth. Not only for the immobile phase such as membrane, but also the protein in cytosol can be measured the parameter in our system – Two photon excitation fluorescence correlation spectroscopy (TPE-FCS). As for these reasons that two photon excitation was the suitable tool to develop a platform for long period of time in observation of cell biology. As a given excitation volume, the diffusion coefficient of molecules due to the Brownian motion into or out of the excitation volume is determined. In our system, the limit concentration of measurement is between sub-nanomolar to sub-micromolar. This is associated to the transfection. After tansfection, the HEK 293 cell started express the green fluorescence protein. However, less than 6 hour after transfection, the cells were at an unstable situation and there are less GFP in the cell. On the other hands, more than 30 hours that too much GFP were expressed in cell even secrete out to the medium. Thus became more background noise to the FCS measurement.
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