Fast and multiplexed superresolution imaging with DNA-PAINT-ERS

Fehmi Civitci,Xiaolin Nan,Lei Wu,Julia Shangguan,Ting Zheng,Kai Tao,Matthew Rames,John Kenison,Ying Zhang,Sadik Esener,Carey Phelps

doi:10.1038/s41467-020-18181-6

Fehmi Civitci, Xiaolin Nan + Show 9 more

Open Access

https://doi.org/10.1038/s41467-020-18181-6

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Abstract

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

Highlights

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed
DNA-PAINT, the localizations arise from reversible hybridizations between a docking strand (DS) oligo immobilized on the target and a complementary, fluorophore-conjugated imager strand (IS) oligo diffusing in solution (Fig. 1, first panel)
We found that ethylene carbonate (EC) accelerated the dehybridization of DS-IS with little impact on the reverse process when added to a DNAPAINT imaging buffer at concentrations as low as 5% (v/v)

Summary

Introduction

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. DNA-PAINT, the localizations arise from reversible hybridizations between a docking strand (DS) oligo immobilized on the target and a complementary, fluorophore-conjugated imager strand (IS) oligo diffusing in solution (Fig. 1, first panel) This imaging scheme simplifies multiplexed SMLM by eliminating the need for photo-switchable fluorophores and allows multiple targets to be DNA-barcoded and imaged sequentially[4]. A practical hurdle to using DNA-PAINT for multiplexed SMLM is the slow imaging speed, with each target taking tens of minutes to hours to complete[4] This is primarily due to relatively slow localization kinetics. It remains unclear how many orthogonal DS-IS pairs like PS3 exist, and other generally applicable methods are necessary for fast and multiplexed DNA-PAINT in biological applications

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