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Research Article

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

Jing Liu1, Tim Hebbrecht2, Toon Brans1, Eef Parthoens3,4,5, Saskia Lippens3,4,5, Chengnan Li6, Herlinde De Keersmaecker1,8, Winnok H. De Vos7, Stefaan C. De Smedt1,8, Rabah Boukherroub6, Jan Gettemans2, Ranhua Xiong1, Kevin Braeckmans1,8( )
Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium
VIB-UGent Center for Inflammation Research, VIB, Ghent B-9000, Belgium
VIB Bioimaging Core Ghent, VIB, Ghent B-9000, Belgium
Department of Biomedical Molecular Biology, Ghent University, Ghent B-9000, Belgium
Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France
Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020 Antwerp, Belgium
Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium
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Graphical Abstract

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.

Keywords

laser-induced photoporationvapor nanobubblelong-term microscopy imagingnanobodyintracellular deliveryliving cell labeling

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Nano Research
Volume 13 Issue 2,
February 2020
Pages 485-495
DOI: 10.1007/s12274-020-2633-z
Cite this article:
Liu J, Hebbrecht T, Brans T, et al. Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation. Nano Research, 2020, 13(2): 485-495. https://doi.org/10.1007/s12274-020-2633-z
Topics:
Cell membranes Cell proliferation Fluorescence microscopyProteins

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Received: 22 October 2019
Revised: 23 December 2019
Accepted: 01 January 2020
Published: 18 January 2020
© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
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