Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS

Anna M. Koester, Malwina Szczepaniak, Xiaolin Nan

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Recent advances in super resolution microscopy have enabled imaging at the 10-20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, has been the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that leads to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5-10 min. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here, we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies).

Original languageEnglish (US)
Article numbere618
JournalCurrent Protocols
Issue number11
StatePublished - Nov 2022


  • multiplexed imaging
  • single-molecule localization microscopy
  • super resolution microscopy

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Health Informatics
  • Medical Laboratory Technology


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