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Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation

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Abstract

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (∼ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

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Acknowledgements

K. B. acknowledges financial support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (No. 648124) and from the Ghent University Special Research Fund (No. 01B04912) with gratitude. J. L. gratefully acknowledges the financial support from the China Scholarship Council (CSC) (No. 201506750012) and the Ghent University Special Research Fund (No. 01SC1416). T. H. and J. G. acknowledges financial support from the Fonds Wetenschappelijk Onderzoek (No. G.0559.16N) and Ghent University (BOF-GOA) (No. BOF13/GOA010). We would like to thank the Centre for advanced light microscopy at Ghent University (Belgium) for the support during the data acquisition and analysis of the colocalization experiments. We would like to thank Mr. Dominique Deresmes for helping to perform the AFM imaging.

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Authors and Affiliations

  1. Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, B-9000, Belgium

    Jing Liu, Toon Brans, Herlinde De Keersmaecker, Stefaan C. De Smedt, Ranhua Xiong & Kevin Braeckmans

  2. Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent, B-9000, Belgium

    Tim Hebbrecht & Jan Gettemans

  3. VIB-UGent Center for Inflammation Research, VIB, Ghent, B-9000, Belgium

    Eef Parthoens & Saskia Lippens

  4. VIB Bioimaging Core Ghent, VIB, Ghent, B-9000, Belgium

    Eef Parthoens & Saskia Lippens

  5. Department of Biomedical Molecular Biology, Ghent University, Ghent, B-9000, Belgium

    Eef Parthoens & Saskia Lippens

  6. Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille, F-59000, France

    Chengnan Li & Rabah Boukherroub

  7. Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020, Antwerp, Belgium

    Winnok H. De Vos

  8. Centre for Advanced Light Microscopy, Ghent University, Ghent, B-9000, Belgium

    Herlinde De Keersmaecker, Stefaan C. De Smedt & Kevin Braeckmans

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  1. Jing Liu
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  2. Tim Hebbrecht
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  3. Toon Brans
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  4. Eef Parthoens
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  9. Stefaan C. De Smedt
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  12. Ranhua Xiong
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Correspondence to Kevin Braeckmans.

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Liu, J., Hebbrecht, T., Brans, T. et al. Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano Res. 13, 485–495 (2020). https://doi.org/10.1007/s12274-020-2633-z

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  • DOI: https://doi.org/10.1007/s12274-020-2633-z

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