Development of the multi-point fluorescence correlation spectroscope and analysis of molecular interaction in living cell

根据细胞内不同的位置，如靠近或远离质膜、细胞核、末端体或细胞核，可以检测到许多物质、pH、离子或氧浓度在活细胞中的不同分布。细胞的微环境也根据细胞的生理条件而改变。本项目的目的是建立一个多点荧光相关光谱测量系统，能够以单分子的灵敏度检测荧光分子的数量浓度，并为活细胞中动态分子相互作用的检测进行基础研究。我们试图构建一个可以使用两种不同荧光强度检测互相关函数的原型系统。然后，检测区域增加到四个位置。最后，我们将位置间移动的时间缩短了至少10毫秒。本课题选择了糖皮质激素受体，它是一种转录调节蛋白，也是TPA刺激后从胞浆到细胞核的转运蛋白。TPA刺激后，细胞核内GR-GFP表达增加，胞浆内GR-GFP表达减少。利用我们的系统（MP-FCS）分析了GR的扩散时间和每个分子的荧光强度。此外，由于该系统可以同时检测两个位置的荧光发射，我们进行了两个检测区域之间的空间相关性分析测量。当分子运动方向相同时，我们将能够检测到图像相关函数中相关幅度的增加。利用该系统可以分析生物分子在活细胞中的动态运动。

Different distribution of many materials, pH, ion, or oxygen concentration can be detected in living cell according to the different position in the cell such as near or away from plasma membrane, nucleus, endsome or mitochondrion. Microenvironment of the cell is also changed according to physiological condition of the cell. The purpose of the project was construction of a multi-point measurement system of Fluorescence Correlation Spectroscopy that could detect number concentration of fluorescent molecule with the single molecule sensitivity and basic research for the detection of dynamic molecular interaction in a living cell.At the first step of the project, we attempted to construct proto type system that could detect cross correlation function using two different fluorescence intensities. Then, detection area was increased to four positions. Finally, we reduced the duration time least 10m sec between moving position to position.Glucocorticoid receptor, that is one of transcriptional regulator protein and traslocating protein from cytosol to nucleus after stimulated by TPA, was selected in the project. We detected the increase number of GR-GFP in nucleus and the decrease that in cytosol by TPA stimulation. The diffusion time and the fluorescence intensity per molecule of GR were analyzed using our system (MP-FCS). Furthermore, we carried out the measurement of spacial correlation analysis between two detection areas because the system could detect fluorescence emission from two positions simultaneously. When the molecules move same direction, we will be able to detect increase of correlation amplitude in image correlation function. By using this system, the dynamic movement of biomolecule will be analyzed in living cell.