Perelman Rebecca T, Church George M, Stein Johannes
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
Harvard Biophysics Program, Harvard University, Boston, MA, USA.
bioRxiv. 2025 Jul 18:2025.07.14.664726. doi: 10.1101/2025.07.14.664726.
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 (NaSO) that overcomes these challenges. Our optimized formulation, combining NaSO with Trolox (SST), effectively preserves docking strand integrity for over 24 hours 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.
DNA-PAINT（纳米级形貌中的点积累）是一种超分辨率显微镜技术，能够通过荧光标记的成像链与互补DNA对接链的瞬时结合实现纳米级成像。成像链可以从一个实际上无限的库中持续补充，这使得DNA-PAINT本质上具有抗光漂白能力。然而，长时间的DNA-PAINT成像受到活性氧（ROS）形成的限制，活性氧会随着时间的推移损坏对接链并减少定位采样。尽管目前最先进的氧清除系统（OSS）可以减轻这种损害，但其酶成分会随着时间的推移而降解，从而降低其性能、稳健性和实用性。在此，我们引入了一种基于亚硫酸钠（NaSO）的简单、无酶氧清除缓冲液，该缓冲液克服了这些挑战。我们优化的配方将NaSO与生育三烯酚（Trolox）相结合（SST），有效地保持对接链完整性超过24小时，并增强了长期成像性能。SST将缓冲液稳定性提高了十倍，易于制备且成本降低了90%以上，为长时间的DNA-PAINT成像提供了一种稳健、经济高效的高性能OSS。
