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As proof-of-principle for generating superresolution structural informationfrom DNA we applied a method of localization microscopy utilizing photoblinkingcomparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13,using a bespoke multicolor single-molecule fluorescence microscope. We used afull-length ~49 kbp {\lambda} DNA construct possessing oligo inserts at eitherterminus allowing conjugation of digoxigenin and biotin at opposite ends fortethering to a glass coverslip surface and paramagnetic microsphererespectively. We observed stochastic DNA-bound dye photoactivity consistentwith dye photoblinking as opposed to binding/unbinding events, evidencedthrough both discrete simulations and continuum kinetics analysis. We analyzeddye photoblinking images of immobilized DNA molecules using superresolutionreconstruction software from two existing packages, rainSTORM and QuickPALM,and compared the results against our own novel home-written software calledADEMS code. ADEMS code generated lateral localization precision values of 30-40nm and 60-70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling,similar to rainSTORM, running more slowly than rainSTORM and QuickPALMalgorithms but having a complementary capability over both in generatingautomated centroid distribution and cluster analyses. Our imaging system allowsus to observe dynamic topological changes to single molecules of DNA inreal-time, such as rapid molecular snapping events. This will facilitatevisualization of fluorescently-labeled DNA molecules conjugated to a magneticbead in future experiments involving newly developed magneto-optical tweezerscombined with superresolution microscopy.

Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes