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Laser confocal microscopy was used to study the real time 3D transport of nanoparticles in tumor and normal tissues under normal hyperthermic conditions. Breast tumor was inoculated to the nude mouse dorsal skin flap window chamber. Rhodamine labeled liposome nanoparticles were injected into the mouse tail vein. The hyperthermic condition was imposed on the tumor through a thermal chamber. Real time 3D images of extravasation of liposome nanoparticles in normal and tumor tissues were reconstructed. Transport of the nanoparticles from the vasculature to the interstitial space was quantified by measuring both spatial and temporal fluorescence intensity variations. Results show that the liposome nanoparticles hardly extravasate into the interstitial of normal tissues under both normal and hyperthermic conditions. The extravasation of the nano-particles in tumor tissues was greatly enhanced by increasing the local temperature to 42°C. The confocal images show that the extravasation of the nanoparticles in tumor is heterogeneous, and depends on the tumor angiogenesis.