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This work attempted to directly observe polymeric nanoparticles in vivo by analytical electron microscopy (AEM) using copper chlorophyll as the contrast agent, based on the experiments concerned with the copper chlorophyll labeled poly-DL-lactide nanoparticles and the in vivo distribution of the polysorbate 80 (T-80)-coated nanoparticles in brain tissues. With the nanoprecipitation method without surfactants, copper chlorophyll is aggregated in the coordinately saturated form and encapsulated by the matrices of nanoparticles, which may ensure the stability of copper chlorophyll during the in vivo experiments. From both morphological information and chemical information, only the labeled nanoparticles with the T-80 coating were directly traced in the brain by AEM. The results not only support the mechanism of endocytosis and/or transcytosis of T-80-coated nanoparticles targeted to the brain but also verify that it is practical to probe polymeric nanoparticles in vivo using AEM together with copper chlorophyll as the contrast agent.