采用共聚焦显微技术中的双光子激发荧光方法获得DHL细胞中5-氨基酮戊酸(5-ALA)代谢原卟啉Ⅸ(PpⅨ)荧光图像。结合Rhodamine123、DioC6(3)和LysoTracker Green三种细胞器荧光探针的使用, 采用双标的标记方法观察DHL细胞中5-ALA代谢PpⅨ在DHL细胞的定位分布, 进而利用Matlab软件对获得的定位的融合荧光图像进行相关系数计算。通过Matlab软件编写程序对获得的定位图像进行边缘检测、提取和分割等处理, 计算获得的线粒体相关系数r=0.564; 内质网相关系数r=0.465; 溶酶体相关系数r=0.366; 可以认为5-ALA代谢PpⅨ在线粒体、内质网、溶酶体三种细胞器区域均有分布, 但PpⅨ在不同细胞器聚集程度存在比较大的差异, 经过3 h的孵育代谢PpⅨ主要积聚于线粒体, 而溶酶体中积聚的最少。研究表明双光子激发荧光可成为定性或定量研究DHL细胞中5-ALA代谢PpⅨ以及PpⅨ在细胞定位的重要方法。

Two-photon excited fluorescence based on confocal laser scanning system was used to study the cellular localization of 5-ALA induced PpⅨ in DHL cells. Fluorescence probes of rhodamine123, DioC6(3) and LysoTracker Green were used to indicate the cellular localization of 5-ALA induced PpⅨ with a double-labeling method, and real-time image processing was carried out to detect the image edges, extract and segment the information of fluorescence images. By using the simulation of Matlab software, we calculated the correlation coefficient between fluorescence probe image and 5-ALA induced PpⅨ image after a 3-hour incubation time, and the results show r=0.564 for mitochondria, r=0.465 for endoplasmic reticulum and r=0.366 for lysosome. It can be seen that 5-ALA induced PpⅨ mainly distributes in mitochondira, but also in endoplasmic reticulum and lysosome, at least in the lysosome. Two-photon excited fluorescence can be a useful method for quantitative or qualitative cellular localization analysis of 5-ALA induced PpⅨ.