激光生物学报, 2005, 14 (2): 139, 网络出版: 2006-05-23  

三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较

Comparison among Three Sets of Fluorescence Microscopy Imaging System in Studying the Intracellular Distribution of Photosensitizers
作者单位
解放军总医院激光科,中国,北京,100853
摘要
目的:对三套荧光显微成像系统在国产新型光敏剂HMME亚细胞定位研究中的应用特点及适用范围进行了比较与评价.方法:分别应用LSCM、CCD、ICCD荧光显微成像系统,选择特异性细胞器荧光探针Rhodamine-123、DIOC6(3)标记细胞内线粒体和内质网.采用细胞器-细胞荧光强度比值法,对 HMME进行单细胞内分布的定性与定量研究.结果:LSCM和CCD成像系统能采集到浓度达到160 μg/ml时的HMME 的荧光图像, 获得荧光探针图像信息显示所标记的细胞内线粒体和内质网平均荧光强度比值(J1/J2值)都明显高于细胞内J1/J2值.而 ICCD成像系统只需HMME浓度为5 μg/ml,荧光图像特点都呈胞浆中荧光强度较高且分布不均,细胞核区荧光较弱的中空现象.ICCD系统对细胞器探针荧光图像在空间分辨上不理想.结论:LSCM与CCD成像系统限于其探测灵敏度,对于弱荧光性光敏剂,适用于其高孵育浓度条件下的亚细胞定位研究.二者获得的结果相一致:孵育24 h,HMME在鼠肺内皮细胞线粒体和内质网有分布而几乎不进入细胞核.ICCD成像系统可不受孵育浓度条件的限制,实现光敏剂极微弱荧光的有效探测,但空间分辨率较低.
Abstract
Objective: to compare three sets of fluorescence microscopy imaging systems with each other applied in study of subcellular localization of domestic new photosensitizer HMME. Methods:three sets of imaging system including LSCM, CCD and ICCD fluorescence microscopy imaging system were applied respectively and two special organelle probes including Rhodamine-123, DiOC6(3)were selected to label mitochondrial and ER. Organelle-cell fluorescence intensity ratio analysis was adopted to study the intracellular distribution of HMME. Results:LSCM and CCD microscopy imaging system could acquire intracellular fluorescence images of HMME at the concentration of 160 μg/ml while ICCD could do it at the concentration of 5 μg/ml; fluorescence intensity in cytoplasm was higher than that in nucleus. Average fluorescence intensity ratio in regions labeled by two organelle probes acquired by the two former systems was markedly higher than that in cells. ICCD microscopy imaging system could not distinguish details of organelle

戴维德, 李晓松, 曾晶, 丁新民, 刘凡光, 顾瑛. 三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较[J]. 激光生物学报, 2005, 14(2): 139. 戴维德, 李晓松, 曾晶, 丁新民, 刘凡光, 顾瑛. Comparison among Three Sets of Fluorescence Microscopy Imaging System in Studying the Intracellular Distribution of Photosensitizers[J]. Acta Laser Biology Sinica, 2005, 14(2): 139.

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