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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Rhodaminel23、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,汁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。
The 5-aminolevulinic acid (5-ALA) metabolites protoporphyrin IX (PpⅨ) fluorescence images of DHL cells were obtained by two-photon excitation fluorescence method in confocal microscopy. Combined with Rhodaminel23, DioC6 (3) and LysoTracker Green three fluorescent probes, the localization of 5-ALA metabolized PpIX in DHL cells in DHL cells was observed by double labeling method, and then the localization of fusion was obtained by using Matlab software Fluorescence image correlation coefficient calculation. The results of edge detection, extraction and segmentation of the obtained positioning images were obtained by Matlab program. The correlation coefficient of r = 0.564, the correlation coefficient of endoplasmic reticulum r = 0.465, and the lysosomal correlation coefficient r = 0.366 It is considered that PpIX of 5-ALA metabolites is distributed in the mitochondria, endoplasmic reticulum and lysosomes. However, there is a big difference in the aggregation of PpⅨ in different organelles. After 3 h of incubation, PpⅨ mainly accumulates in the mitochondria, Lysosomes accumulate least. Studies have shown that two-photon excitation fluorescence can be a qualitative or quantitative study of DHL cells 5-ALA metabolism of PpⅨ and PpIX important method of cell location.