High-throughput single molecule microscopy with adaptable spatial resolution using exchangeable oligonucleotide labels

¹ Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, The Netherlands
² Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands

Published online: 22 September 2025

Abstract

Super-resolution microscopy based on the localization of single molecules facilitates the visualization of cellular structures at a resolution approaching the molecular level. However, their low-throughput nature hampers their applicability in biomolecular research and screening. Here, we propose an efficient workflow, starting with the scanning of large areas using fast fluctuation-based imaging, followed by single-molecule localization microscopy of selected cells. We exploit the versatility of DNA oligo hybridization kinetics with DNA-PAINT probes to tailor the fluorescent blinking toward high-throughput and high-resolution imaging. Additionally, we employ super-resolution optical fluctuation imaging (SOFI) to analyze statistical fluctuations in the DNA-PAINT binding kinetics, thereby tolerating much denser blinking and facilitating accelerated imaging speeds. We demonstrate 30–300-fold faster imaging of different cellular structures compared to conventional DNA-PAINT imaging, albeit at a lower resolution. Notably, by tuning the image medium and data processing, we can flexibly switch between high-throughput SOFI (scanning an FOV of 0.65 mm × 0.52 mm within 4 min of total acquisition time) and super-resolution DNA-PAINT microscopy and thereby demonstrate that combining DNA-PAINT and SOFI enables one to adapt image resolution and acquisition time based on the imaging needs. We envision this approach to be especially powerful when combined with multiplexing and 3D imaging.