A Simple, Ultrastable, and Cost‐Effective Oxygen‐Scavenging System for Long‐Term DNA‐PAINT Imaging

TL;DR

A new, low-cost oxygen-scavenging system improves DNA-PAINT microscopy by preserving DNA integrity and enabling long-term high-resolution imaging.

Contribution

An enzyme-free, cost-effective oxygen scavenging buffer using sodium sulfite and Trolox enhances DNA-PAINT performance and stability.

Findings

The new system preserves docking strand integrity for over 24 hours.

The buffer improves stability tenfold and reduces costs by over 90%.

It enables faster and higher-precision localizations in DNA-PAINT imaging.

Abstract

DNA‐PAINT (Points Accumulation in Nanoscale Topography) is a super‐resolution microscopy technique capable of nanoscale imaging through the transient binding of fluorescently labeled imager strands to complementary DNA docking strands. Imager strands can be continuously replenished from an effectively infinite pool, making DNA‐PAINT inherently resistant to photobleaching. However, extended DNA‐PAINT imaging is limited by the formation of reactive oxygen species (ROS), which damage docking strands and reduce localization sampling over time. Although the state‐of‐the‐art oxygen‐scavenging system (OSS) can mitigate this damage, its enzymatic components degrade over time, reducing its performance, robustness, and utility. Here, we introduce a simple, enzyme‐free oxygen scavenging buffer based on sodium sulfite (Na2SO3) that overcomes these challenges. Our optimized formulation, combining Na2SO3 with Trolox (SST), effectively preserves docking strand integrity for over 24 h and enhances long‐term imaging performance. SST improves buffer stability tenfold, is easy to prepare and reduces costs by more than 90%, providing a robust, cost‐effective, high‐performance OSS for extended DNA‐PAINT imaging.

A simple, enzyme‐free, and cost‐effective oxygen scavenging system enhances DNA‐PAINT super‐resolution microscopy by increasing photostability, minimizing docking strand degradation, and enabling faster, high‐precision localizations. Its long‐term stability supports extended imaging, yielding improved spatial resolution and image quality. This robust system provides a foundation for advancing a wide range of DNA‐based multiplexed imaging techniques.

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