Herein, we demonstrate the potential of ultrabright fluorescent silica-coated organic nanocrystals for two-photon in vivo imaging. These unique nanoparticles (NPs) containing a crystalline core of small push-pull dipolar dye are specifically designed to exhibit two-photon absorption for 2 fluorescence imaging. The NPs can be easily functionalized using click chemistry in pure water, with preservation of the organic core. A novel small-volume DLS technique was used to evaluate the effect of PEGylation on the colloidal stability of the NPs in complex media containing salts and proteins, mimicking the composition of blood serum. The potential of these bright red-emitting NPs for two-photon fluorescence imaging is demonstrated both in vitro and in vivo.

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