| Objective To compare three sets of fluorescence microscopy imaging systems with each other applied in study of subcellular localization of domestic new photosensitizer HMME. To establish a new quantitive analytical method of subcellular localization and new real-time detecting method of photodamage sites. To discuss influence factors of subcellular distribution of photosensitizers.Methods & Results Three sets of imaging system including LSCM imaging system, CCD fluorescence microscopy imaging system and ICCD fluorescence microscopy imaging system were applied respectively and organelle-cell fluorescence intensity ratio analysis was adopted to study the intracellular distribution of HMME. To study the influence of cell type, incubating time and combination form of HMME with different kinds of serum protein on intracellular distribution of HMME. Fluorescence images of ROS probe H2DCF-DA and organelle probes were detected and analyzied to determine production sites of ROS in single cell. Morphology transformation of organelle probes' fluorescence in single cell before, during and after irradiation was observed to investigate subcellular localization of photodamage sites and dynamic processs of photodamage.The results showed: (1) LSCM and CCD microscopy imaging system could acquire intracellular fluorescence images of HMME at the concentration of 160μg/ml while ICCD could do it at the concentration of 5μg/ml, fluorescence intensity in cytoplasma was higher than that in nucleus. Average fluorescence intensity ratio in regions labeled by fours 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 probes' fluorescence images. (2) With incubating time prolonging, J|/J2 ofA549 cells' four organelles were increasing , and J1/J2 of endothelial cells'lysosome was increasing, while that of three other organelles including Golgi, ER and mitochondria was decreasing; the increasing of J1/ J2 of A549 cells'lysosome was higher than that of endothelial cells'. (3) Comparing cells incubating for 2h with that for 12h, Ji/J2 of HMME of four organelles in non-serum group were all increasing, it was significant for three organelles including mitochondria, ER and Golgi; Lysosome's J1/J2 of HMME in Alb group was increasing comparative significantly than that of the other three organelles; Among the four organelles in LDL group, Lysosome's J1/J2 of HMME was increasing most significantly, which of other organelles' increasing extents were apparently greater than that in Alb group. (4) Fluorescence intensity of the whole cell was decreased by 26.4% after irradiation. According to every organelle's average fluorescence intensity ratio before irradiation, which of mitochondria, lysosome, ER and Golgi were decreased by 31.1%, 23.4%, 5.8% and 5% after irradiation separately enhancing fluorescence of DCF was detected in mitochondria with HMME or not after irradiation. (5) Difference of Rhodamine-123's fluorescence images was significant before and after photodamage.The formal character of them has changed obviously. Rhodamine-123 redistributed in nucleus after irradiation.Conclusions (1) To be limited to their detecting sensitivity, LSCM and CCD microscopy imaging system are applicable to subcellular localization of weak fluorescent photosensitizers at the high incubating concentration. The experiment results acquired by both of them are same with each other. Incubating for 24h, HMME mainly distributes in cytoplasm including mitochondria, lysosome, ER and Golgi; little is in nucleus. ICCD microscopy imaging system established by us can effectively detect hypofluorescence of photosensitizers without limitation of incubation concentration. (2) Cell type and incubating time are important influence factors of subcellular distribution of HMME. With incubating time prolonging, four organelles' ability to absorb HMME is enhancing in A549 cells, especially does lysosome; three organelles' ability to absorb HMME is weakening in endothelia...
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