Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish |
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Authors: | Marcelina Cardoso Dos Santos Ingrid Colin Gabriel Ribeiro Dos Santos Kimihiro Susumu Michaël Demarque Igor L Medintz Niko Hildebrandt |
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Affiliation: | 1. Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CNRS, CEA, Orsay Cedex, 91405 France;2. Institut des Neurosciences Paris-Saclay, Université Paris-Saclay, CNRS, Gif-sur-Yvette, 91190 France;3. Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, D.C., 20375 USA
KeyW Corporation, Hanover, MD, 21076 USA;4. Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, D.C., 20375 USA |
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Abstract: | 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. |
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Keywords: | Förster resonance energy transfer imaging lanthanides multiplexing quantum dots |
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