Deep Learning and Single-Molecule Localization Microscopy Reveal Nanoscopic Dynamics of DNA Entanglement Loci

Abstract

Understanding molecular dynamics at the nanoscale remains challenging due to limitations in the temporal resolution of current imaging techniques. Deep learning integrated with Single-Molecule Localization Microscopy (SMLM) offers opportunities to probe these dynamics. Here, we leverage this integration to reveal entangled polymer dynamics at a fast time scale, which is relatively poorly understood at the single-molecule level. We used Lambda DNA as a model system and modeled their entanglement using the self-avoiding wormlike chain model, generated simulated localizations along the contours, and trained the deep learning algorithm on these simulated images to predict chain contours from sparse localization data. We found that the localizations are heterogeneously distributed along the contours. Our assessments indicated that chain entanglement creates local diffusion barriers for switching buffer molecules, affecting the photoswitching kinetics of fluorescent dyes conjugated to the DNA molecules at discrete DNA segments. Tracking these segments demonstrated stochastic and subdiffusive migration of the entanglement loci. Our approach provides direct visualization of nanoscale polymer dynamics and local molecular environments previously inaccessible to conventional imaging techniques. In addition, our results suggest that the switching kinetics of the fluorophores in SMLM can be used to characterize nanoscopic local environments.