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Site-specifically-labeled antibodies for super-resolution microscopy reveal in situ linkage errors

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posted on 02.07.2021, 15:48 authored by Susanna M Früh, Ulf Matti, Philipp R Spycher, Marina Rubini, Sebastian Lickert, Thomas Schlichthaerle, Ralf Jungmann, Viola Vogel, Jonas Ries, Ingmar SchoenIngmar Schoen
The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody's Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.


Holcim Science Foundation

The European Research Council (ERC CoG-724489)

The European Research Council (ERC StG-680241)

DFG (S.M.F., 397660978)

DFG (JU 2957/1-1)

DFG (SFB 1032/A11)

AiF (S.M.F., 20436 N)

QBM graduate school

Center for Nano-Science

European Molecular Biology Laboratory

ETH Zurich

Max Planck Society/Foundation




The original article is available at

Published Citation

Früh SM, et al. Site-specifically-labeled antibodies for super-resolution microscopy reveal in situ linkage errors. ACS Nano. 2021

Publication Date

29 Jun 2021

PubMed ID



  • Irish Centre for Vascular Biology
  • School of Pharmacy and Biomolecular Sciences

Research Area

  • Vascular Biology
  • Biomaterials and Regenerative Medicine


American Chemical Society (ACS)


  • Published Version (Version of Record)