激光共聚焦显微镜在细胞内ROS研究中的激光干扰问题

目的 对激光共聚焦显微镜在细胞内ROS研究中的激光干扰问题进行探讨.方法 ROS探针标记ECV-304及BAW264.7细胞后,激光共聚焦显微镜488nm波长检测器分别进行连续和间隔观测1200s,比较两种观测方式下细胞荧光强度的变化;外源性加入H2O2,证实间隔观测时系统的灵敏性.结果 在连续观测方式下,细胞荧光强度短时间内显著增强,改为间隔观测后细胞荧光强度变化缓慢,但维持在低水平且在外源性加入H2O2后细胞荧光强度迅速增强.结论 激光共聚焦显微镜激发激光可引起细胞大量产生ROS,对检测系统造成干扰,采用间隔观测的方法可显著改善系统自身的这种缺陷,并能保持系统的检测灵敏性.

Laser interference in testing of intracellular reactive oxygen species with laser confocal microscope

Objective To discuss laser interference when applying laser confocal microscope to test intracellutar reactive oxygen species.Methods ECV-304 cells and RAW264.7 cells were labelled with ROS molecular probe H2 DCFDA. The fluorescent intensity of the labelled cells were tested continuously and discontinuously, respectively. The sensitivity of the cliscontinous observation was con_flnned by adding H2O2 exogenously. Results By continuous observation, the fluorescent intensity of the tested cells markedly increased in short period. However, the changes of the fluorescent intensity of the tested cells were slow and the fluorescent intensity maintained low level when discontinuous observation was applied. The fluorescent intensity increased rapidly by discontinous observation when H2 O2 was added exogenously. Conclusion The 488nm laser could interfere the outcomes of endocelluar ROS, and discontinuous observation could mend this limitation.