激光共聚焦显微成像技术对亚细胞位点光动力损伤的动态观察

Adopting laser scanning confocal microscopy in dynamic observation of photodamage of subcellular sites

目的探讨应用激光共聚焦显微成像技术对光动力效应所致亚细胞位点损伤进行动态观察的可行性。方法实验分为HMME+Rhodamine123组、单加HMME组、单加Rhodamine123组和单纯细胞组,后3组作为对照组。传代培养鼠肺毛细血管内皮细胞,将血卟啉单甲醚(HMME)与内皮细胞共同孵育24h后,加入细胞探针Rhodamine123对线粒体进行染色。应用激光共聚焦显微镜并采用细胞器细胞荧光强度比值法对HMME进行亚细胞定位,随后采集Rhodamine123的荧光图像动态序列。结果HMME+Rhodamine123组的Rhodamine123荧光图像在光照过程中逐渐发生变化:典型的线粒体形态特征逐渐消失,并伴有荧光强度下降,荧光在胞浆内趋于弥散分布,细胞核内出现Rhodamine123的荧光;而单加Rhodamine123组的Rhodamine123荧光图像未发生明显变化。结论应用激光共聚焦显微成像技术能实现亚细胞水平光敏损伤位点的动态光学检测,线粒体和核膜可能是HMME介导的光动力损伤亚细胞位点。

Objective To dynamically observe photodamage of subcellular sites by use of laser scanning confocal microscopy （LSCM）. Methods The samples were divided into four groups. Murine lung endothelial cells were subcuhured and incubated with HMME for 24 hours, Then the cells were stained with rhodamine-123 for demonstration of mitochondria. LSCM was applied and organelle-cell fluorescence intensity ratio analysis was adopted to study the intracellular distribution of HMME. Then dynamic fluorescence images sequence of rhodamine-123 was collected. Results Rhodamine-123＇s fluorescence images of cell sample with HMME was changed gradually during irradiation： the typical characteristic of mitochondria disappeared gradually with decreasing fluorescence intensity. The fluorescence of rhodamine-123 was diffused and distributed in nuclear, while rhodamine-123＇s fluorescence images of cell sample without HMME was not changed, Conclusion Mitochondria and nucleus are photodamage sites by HMME-PDT; LSCM can be applied in dynamic observation of photodamage of subcelluar sites.