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

Cells. 2019 Jan 14;8(1):48. doi: 10.3390/cells8010048.


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.

Keywords: DNA-PAINT; fluorescent proteins; linkage error; microfluidics; molecular localization; multi-color imaging; multiplexing; nanobodies; single domain antibodies (sdAb); super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Chromatin / chemistry
  • Chromatin / ultrastructure
  • DNA / chemistry
  • Golgi Apparatus / chemistry
  • Golgi Apparatus / ultrastructure
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / immunology
  • Microscopy, Fluorescence / methods*
  • Mitochondria / chemistry
  • Mitochondria / ultrastructure
  • Single Molecule Imaging / methods*
  • Single-Domain Antibodies / immunology*


  • Chromatin
  • Luminescent Proteins
  • Single-Domain Antibodies
  • DNA