Fluorescence Image Analysis for Immune Respnses

用氩离子激光（488 nm）和氦镉激光（325 nm）共聚焦荧光显微镜研究了大鼠嗜碱性白血病细胞（RBL-2 H3细胞）内钙信号的空间异质性。在抗原（DNP_7 - BSA）刺激下，RBL-2 H3细胞的Fluo-3荧光强度增加，但延迟时间不同。Fluo-3荧光强度的时间依赖性曲线与Fura-2的顺序荧光比率图像的模式非常相似，Fura-2的顺序荧光比率图像用于通过常规荧光显微镜测量单个RBL-2 H3细胞中的[Ca^<2+>]_i。共聚焦荧光显微镜观察结果显示，Fluo-3荧光强度存在空间异质性，提示RBL-2 H3细胞内[Ca^<2+>] i存在空间异质性。也就是说，结果表明，在RBL-2 H3细胞中，钙信号首先在伪足处瞬时发生，然后信号转移到细胞的中央部分。此外，从共负载Hoechst 33342（DNA特异性探针）的He-Cd激光激发的荧光图像中，发现细胞核的荧光图像与上述钙信号的荧光图像非常相似。这表明受体介导的钙信号不仅传递到细胞质，而且还传递到细胞核。

A confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm) , was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia cells (RBL-2H3 cells). After the stimulation of antigen (DNP_7 - BSA), fluo-3 fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Timedependent profiles of the fluo-3 fluorescence intensities resembled closely the patterns of the sequential fluorescence ratio images of fura-2, which were used to measure [Ca^<2+>]_i in individual RBL-2H3 cells by a conventional fluorescence microscope. The present results by the confocal fluorescence microscope showed spatial heterogeneities of fluo-3 fluorescence intensities, suggesting the exsstence of the spatial heterogeneity of[Ca^<2+>]_i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopods in RBL-2H3 cells and then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (DNA-specific probe) which were excited by a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.