Evaluation of direct grafting strategies via trivalent anchoring for enabling lipid membrane and cytoskeleton staining in expansion microscopy

Gang Wen, Marisa Vanheusden, Aline Acke, Donato Valli, Robert Neely, Volker Leen, Johan Hofkens

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Super-resolution fluorescence microscopy is a key tool in the elucidation of biological fine structures, providing insights into the distribution and interactions of biomolecular complexes down to the nanometer scale. Expansion microscopy is a recently developed approach for achieving nanoscale resolution on a conventional microscope. Here, biological samples are embedded in an isotropically swollen hydrogel. This physical expansion of the sample allows imaging with resolutions down to the tens-of-nanometers. However, because of the requirement that fluorescent labels are covalently bound to the hydrogel, standard, small-molecule targeting of fluorophores has proven incompatible with expansion microscopy. Here, we show a chemical linking approach that enables direct, covalent grafting of a targeting molecule and fluorophore to the hydrogel in expansion microscopy. We show application of this series of molecules in the antibody-free targeting of the cell cytoskeleton and in an example of lipid membrane staining for expansion microscopy. Furthermore, using this trivalent linker strategy, we demonstrate the benefit of introducing fluorescent labels post-expansion by visualizing an immunostaining through fluorescent oligonucleotide hybridization after expanding the polymer. Our probes allow different labeling approaches that are compatible with expansion microscopy.
Original languageEnglish
Pages (from-to)7860-7867
Number of pages8
JournalACS Nano
Issue number7
Early online date16 Mar 2020
Publication statusPublished - 28 Jul 2020

Bibliographical note

Funding Information:
Financial support from the Flemish government through long-term structural funding Methusalem (CASAS2, Meth/15/04) to J.H., from CSC through a fellowship to G.W. (201806210078), from FWO through fellowships to M.V. (1S62318N) and A.A. (1193720N), from KULeuven research fund through ID-N project IDN/19/039 and from EPSRC through grant number EP/Nr020901/1 to R.K.N is gratefully acknowledged. The authors would like to thank R. Nuyts and A. De Weerdt for technical support as well as S. Rocha and W. Vandenberg for the fruitful discussions

Publisher Copyright:
Copyright © 2020 American Chemical Society.

Copyright 2020 Elsevier B.V., All rights reserved.


  • expansion microscopy
  • trifunctional linker
  • lipid membranes
  • actin filaments
  • post-expansion labeling


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