Nanobody detection of standard fluorescent proteins enables multi-target DNA-PAINT with high resolution and minimal displacement errors

Shama Sograte-Idrissi, Nazar Oleksiievets, Sebastian Isbaner, Mariana Eggert-Martinez, Jörg Enderlein, Roman Tsukanov, Felipe Opazo

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

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.

Original languageEnglish
Article number48
JournalCells
Volume8
Issue number1
DOIs
StatePublished - 1 Jan 2019
Externally publishedYes

Keywords

  • DNA-PAINT
  • Fluorescent proteins
  • Microfluidics
  • Molecular localization
  • Multi-color imaging
  • Nanobodies
  • Single domain antibodies (sdAb)
  • Super-resolution microscopy
  • linkage error
  • multiplexing

ASJC Scopus subject areas

  • General Medicine

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