Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors
Shama Sograte-Idrissi 1,2,3,†, Nazar Oleksiievets 4,†, Sebastian Isbaner 4
, Mariana Eggert-Martinez 1,2,3, Jörg Enderlein 4, Roman Tsukanov 4,* and Felipe Opazo 1,2,*
, Mariana Eggert-Martinez 1,2,3, Jörg Enderlein 4, Roman Tsukanov 4,* and Felipe Opazo 1,2,*
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)
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-TextKeywords:
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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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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