Minimally Invasive DNA-Mediated Photostabilization for Extended Single-Molecule and Super-resolution Imaging

Abstract

Photobleaching of fluorescence labels poses a major limitation in single-molecule and super-resolution microscopy. Conventional photostabilization methods, such as oxygen removal and addition of high concentrations of photostabilization additives, often require careful fluorophore selection and can disrupt the biological environment. To address these limitations, we developed a modular and minimally invasive photostabilization approach that utilizes DNA-mediated delivery of a photostabilizer directly to the imaging site. Under lower excitation intensities, the DNA-mediated strategy outperformed solution-based approaches, achieving efficient photostabilization at significantly lower additive concentrations. However, at higher excitation intensities, the stability of a single photostabilizer molecule became the limiting factor. To overcome this and reduce the loss of localizations in DNA-PAINT experiments we have also implemented a recovery scheme where the photostabilizer is continuously replenished at the imaging site. We further extended the approach to cell imaging, demonstrating improved localization rate and precision in 3D-DNA PAINT measurements. DNA-mediated photostabilization offers a promising solution for imaging applications where high additive concentrations are prohibited. Its modularity enables adaptation to various imaging schemes and ultimately expands the repertoire of fluorophores suitable for single-molecule and super-resolution imaging.