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Afterglow optical agents, which emit light long after cessation of excitation, hold promise for ultrasensitive in vivo imaging because they eliminate tissue autofluorescence. However, afterglow imaging has been limited by its reliance on inorganic nanoparticles with relatively low brightness and short-near-infrared (NIR) emission. Here we present semiconducting polymer nanoparticles (SPNs) <40 nm in diameter that store photon energy via chemical defects and emit long-NIR afterglow luminescence at 780 nm with a half-life of ∼6 min. In vivo, the afterglow intensity of SPNs is more than 100-fold brighter than that of inorganic afterglow agents, and the signal is detectable through the body of a live mouse. High-contrast lymph node and tumor imaging in living mice is demonstrated with a signal-to-background ratio up to 127-times higher than that obtained by NIR fluorescence imaging. Moreover, we developed an …