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With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, 2,,7,-dichlorofluorescin diacetate (DCFH-DA) and Fluo 3-AM, we simultaneously observed arsenic trioxide (As2O3)-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration [Ca2+]i within human skin squamous carcinoma cells (Colo-16 cells). Our results indicated that As2O3 induced [Ca2+]i elevation and ROS production within Colo-16 cells, and both [Ca2+]i elevation and ROS production were involved in the apoptosis of Colo-16 cells. These results suggested that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multi-parameter observation of As2O3- induced apoptosis at the single cell level.
Key words: Two-photon laser scanning microscopy, confocal laser scanning microscopy, human skin squamous carcinoma cells (Colo-16 cells), arsenic trioxide, apoptosis.