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Time‐Gated FRET Nanoprobes for Autofluorescence‐Free Long‐Term In Vivo Imaging of Developing Zebrafish

The zebrafish is an important vertebrate model for disease, drug discovery, toxicity, embryogenesis, and neuroscience. In vivo fluorescence microscopy can reveal cellular and subcellular details down to the molecular level with fluorescent proteins (FPs) currently the main tool for zebrafish imaging. However, long maturation times, low brightness, photobleaching, broad emission spectra, and sample autofluorescence are disadvantages that cannot be easily overcome by FPs. Here, a bright and photostable terbium‐to‐quantum dot (QD) Förster resonance energy transfer (FRET) nanoprobe with narrow and tunable emission bands for intracellular in vivo imaging is presented. The long photoluminescence (PL) lifetime enables time‐gated (TG) detection without autofluorescence background. Intracellular four‐color multiplexing with a single excitation wavelength and in situ assembly and FRET to mCherry demonstrate the versatility of the TG‐FRET nanoprobes and the possibility of in vivo bioconjugation to FPs and combined nanoprobe‐FP FRET sensing. Upon injection at the one‐cell stage, FRET nanoprobes can be imaged in developing zebrafish embryos over seven days with toxicity similar to injected RNA and strongly improved signal‐to‐background ratios compared to non‐TG imaging. This work provides a strategy for advancing in vivo fluorescence imaging applications beyond the capabilities of FPs.