Trifunctional linkers enable improved visualization of actin by expansion microscopy

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

ABSTRACT Expansion Microscopy (ExM) revolutionized the field of super-resolution microscopy by allowing for subdiffraction resolution fluorescence imaging on standard fluorescence microscopes. However, it has been found that it is hard to visualize actin filaments efficiently using ExM. To improve actin imaging, multifunctional molecules have been designed, however, with moderate success. Here, we present optimized methods for phalloidin conjugate grafting that have high efficiency for both cellular and tissue samples. Our optimized strategy improves anchoring and signal retention by ∼10 times. We demonstrate the potential of trifunctional linkers (TRITON) for actin imaging in combination with immunolabeling using different ExM protocols. 10x ExM of actin labeled with TRITON enabled us to visualize the periodicity of actin rings in cultured hippocampal neurons and brain slices by Airyscan confocal microscopy. Thus, TRITON linkers provide an efficient grafting method, especially in cases where the concentration of target-bound monomers is insufficient for high-quality ExM.

Authors 6

1. Gang Wen

   University of Würzburg · KU Leuven

   Affiliation as printed

   Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

   Department of Chemistry, KU Leuven, Leuven, 3001, Belgium

   Department of Chemistry, KU Leuven, Leuven, 300, Belgium

2. Matthew Domenic Lycas

   University of Würzburg

   Affiliation as printed

   Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

3. Yuqing Jia Aachen Department of Cell and Chemical Biology

   Leiden University Medical Center

   Affiliation as printed

   Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, Netherlands

   Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2 ZC, Leiden, Netherlands

4. Volker Leen

   Décision et Information pour les Systèmes de Production

   Affiliation as printed

   Chrometra Scientific, Kortenaken 3470, Belgium

   Chrometra Scientific, Kortenaken 370, Belgium

5. Markus Sauer corresponding

   University of Würzburg

   Affiliation as printed

   Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

   Rudolf Virchow Center, Research Center for Integrative and Translational Bioimaging, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany

6. Johan Hofkens corresponding

   Max Planck Institute for Polymer Research · KU Leuven

   Affiliation as printed

   Department of Chemistry, KU Leuven, Leuven, 3001, Belgium

   Max Planck Institute for Polymer Research, 55128 Mainz, Germany

   Department of Chemistry, KU Leuven, Leuven, 300, Belgium