Base pair distance analysis in single DNA molecule by direct stochastic optical reconstruction microscopy

Precise fluorescence imaging of single λ-DNA molecules for base pair distance analysis requires a superresolution technique, as these distances are on the order of diffraction limit. Individual λ-DNA molecules intercalated with the fluorescent dye YOYO-1 were investigated at subdiffraction spatial resolution by direct stochastic optical reconstructionmicroscopy (dSTORM). Various dye-to-DNA base pair ratios were imaged by photoswitching YOYO-1 between the fluorescent state and the dark state using two laser sources. The acquired images were reconstructed into a super-resolution image by applying Gaussian fitting to the centroid of the point spread function. By measuring the distances between localized fluorophores, the base pair distances in single DNA molecules for dye-to-DNA base pair ratios of 1:50, 1:100, and 1:500 were calculated to be 17.1±0.8 nm, 34.3±2.2 nm, and 170.3±8.1 nm, respectively, which were in agreement with theoretical values. These results demonstrate that intercalating dye in a single DNA molecule can be photoswitched without the use of an activator fluorophore, and that super-localization precision at a spatial resolution of ~17 nm was experimentally achieved.