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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

1
Institute of Neuro- and Sensory Physiology, University Medical Center Göttingen, 37073 Göttingen, Germany
2
Center for Biostructural Imaging of Neurodegeneration (BIN), University of Göttingen Medical Center, 37075 Göttingen, Germany
3
International Max Planck Research School for Molecular Biology, Göttingen, Germany
4
Third Institute of Physics-Biophysics, Georg August University, 37077 Göttingen, Germany
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Cells 2019, 8(1), 48; https://doi.org/10.3390/cells8010048
Received: 14 December 2018 / Revised: 10 January 2019 / Accepted: 11 January 2019 / Published: 14 January 2019
(This article belongs to the Special Issue Development and Challenges in Microscopy for Cellular Imaging)
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Abstract

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time. View Full-Text
Keywords: nanobodies; super-resolution microscopy; multi-color imaging; fluorescent proteins; microfluidics; DNA-PAINT; molecular localization; single domain antibodies (sdAb); multiplexing; linkage error nanobodies; super-resolution microscopy; multi-color imaging; fluorescent proteins; microfluidics; DNA-PAINT; molecular localization; single domain antibodies (sdAb); multiplexing; linkage error
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Sograte-Idrissi, S.; Oleksiievets, N.; Isbaner, S.; Eggert-Martinez, M.; Enderlein, J.; Tsukanov, R.; Opazo, F. Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors. Cells 2019, 8, 48.

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