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A simple, versatile method for GFP-based super-resolution microscopy via nanobodies

Abstract

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.

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Figure 1: Single-molecule nanoscopy by detection of GFP via nanobodies.
Figure 2: Single-molecule nanoscopy of proteins from a budding yeast GFP-fusion construct library.

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Acknowledgements

This work was supported by a Marie Curie Intra-European Fellowship (J.R.) the National Center for Competence in Biomedical Imaging and the National Center for Competence in Research 'Neural Plasticity and Repair'. We thank Y. Barral and members of the Barral laboratory for useful discussions, help with yeast strains and access to the yeast genomic GFP-fusion library, R. Dechant for help with elutriation and M. Stoeber for help with Amaxa transfection.

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H. Ewers conceived of the project; J.R., C.K., E.P. and H. Ewers designed experiments; J.R., C.K., E.P., H. Eghlidi & H. Ewers performed and analyzed experiments; H. Ewers and J.R. wrote the paper.

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Correspondence to Helge Ewers.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5 and Supplementary Note (PDF 3815 kb)

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Ries, J., Kaplan, C., Platonova, E. et al. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat Methods 9, 582–584 (2012). https://doi.org/10.1038/nmeth.1991

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