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Nano Research

Article Title

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

Authors

Jing Liu, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Tim Hebbrecht, Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium
Toon Brans, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Eef Parthoens, 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
Saskia Lippens, 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
Chengnan Li, Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France
Herlinde De Keersmaecker, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium
Winnok H. De Vos, Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, 2020 Antwerp, Belgium
Stefaan C. De Smedt, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium
Rabah Boukherroub, Univ. Lille, CNRS, Centrale Lille, ISEN, Univ. Valenciennes, UMR 8520-IEMN, Lille F-59000, France
Jan Gettemans, Department of Biomolecular medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent B-9000, Belgium
Ranhua Xiong, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium
Kevin Braeckmans, Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent B-9000, Belgium Centre for Advanced Light Microscopy, Ghent University, Ghent B-9000, Belgium

Keywords

laser-induced photoporation, vapor nanobubble, long-term microscopy imaging, nanobody, intracellular delivery, living cell labeling

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.

Graphical Abstract

Publisher

Tsinghua University Press

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