The imaging system to study the signal transduction in living cells

光学显微镜在基础生物学研究中的应用发生了一场革命。与光学显微镜结合使用的几项新技术为我们提供了一种观察和测量活细胞中事件的新可能性。因此，在本研究项目中，我们试图准备一种新的光学显微镜系统来研究免疫细胞中的信号转导。用新型共聚焦荧光显微镜，分别用Ar离子激光(488 Nm)和He-Cd激光(325 Nm)研究了大鼠嗜碱性白血病细胞(RBL-2H3)、B淋巴细胞和T淋巴细胞钙信号的空间异质性。这些细胞的共聚焦荧光图像显示，受体介导的钙信号不仅转移到细胞质，而且转移到细胞核。该共聚焦荧光显微镜用于观察肥大细胞的胞吐分泌过程，并与荧光分子旋转体9-二氰菊酯(DCVJ)联合使用。众所周知，DCVJ是一种独特的荧光染料，它通过减少分子内的旋转来增加其量子产率。然后，用化合物48/80刺激负载DCVJ的大鼠腹膜肥大细胞，并将其荧光图像与负载Fluo-3的肥大细胞的荧光钙图像进行比较。随着细胞内游离钙离子浓度的短暂升高，DCVJ荧光在细胞质中显著增加，并在细胞核周围形成环状结构，表明该染料可能结合到构成细胞骨架结构的蛋白质上。此外，在细胞松弛素D的存在下，DCVJ的荧光强度的增加大部分被阻断，而在加入化合物48/80后，Fluo-3的荧光强度仍然增加。因此，该成像系统成为研究免疫反应中信号转导的有力工具。

There is a revolution in the use of light microscopy in basic biological research. Several new technologies, used in conjunction with the light microscope, give us a new possibility of seeing and measuring events in the living cell. Thus, in this research project, we have tried to prepare a new system of light microscopy to study signal transduction in immunological cells. By our new confocal fluorescence microscope with an argon-ion laser ( 488 nm) and a He-Cd laser (325 nm) we were able to study spatial heterogeneity of the calcium signals in rat basophilic leukemia cells (RBL-2H3), B lymphocytes and T lymphocytes. Confocal fluorescence images of these cells showed that th e receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus. This confocal fluorescence microscope was used to the exocytotic secretory process of mast cells in combination with an fluorescent molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ). DCVJ is known to be an unique fluorescent dye which increases its qantum yield with decreasing intramolecular rotation. Then, DCVJ-loaded peritoneal rat mast cells were stimulated with compound 48/80 and their fluorescence images were compared with fluorescence calcium images of fluo-3-loaded mast cells. Subsequent to transient increases in intracellular free calcium ion concentration, DCVJ fluorescence increased dramaticaly in the cytoplasm and formed a ring-like structure around the nucleus, suggesting the possibility that the dye bound to the proteins composing the cytoskeletal architecture. Furthermore, the increases of DCVJ fluorescence intensities were mostly blocked in the presence of cytochalasin D. However, fluo-3 fluorescence intensities still increased after addition of compound 48/80. Thus, this imaging system became a powerful tool to study the signal transduction in t he immune responses.