Efficient cell surface labelling of live zebrafish embryos: wash-free fluorescence imaging for cellular dynamics tracking and nanotoxicity evaluation

Imaging the dynamics and behaviors of plasma membranes is at the leading edge of life science research. We report here the development of a universal red-fluorescent probe Chol–PEG–Cy5 for wash-free plasma membrane labelling both in vitro and in vivo. In aqueous solutions, the fluorescence of Chol–PEG–Cy5 is significantly quenched due to the intermolecular resonance energy transfer (RET) between neighbouring Cy5 moieties; however, upon membrane anchoring, the probes undergo lateral diffusion in lipid bilayers, resulting in weakened RET and turn-on fluorescence emission. We demonstrate that Chol–PEG–Cy5 enables rapid, stable and high-quality in vitro cell surface imaging in a variety of mammalian cells. Additionally, with the assistance of three-dimensional (3D) image reconstruction, we achieve for the first time the whole-mount in situ fluorescence imaging of the epidermal cell surfaces of live zebrafish embryos, which cannot be realized by conventional plasma membrane probes due to the presence of the surface-covering mucus barrier. This novel technique encourages us to track the cellular dynamics of the epidermis during embryonic development with 3D visualization. Moreover, we also develop a new method to evaluate the epidermal toxicity of nanomaterials (e.g., gold nanoparticles and graphene oxide nanosheets) toward zebrafish embryos using this fluorescent probe.