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

Precise fluorescence imaging of single l-DNA molecules for base pair distance analysis requires a superresolution technique, as these distances are on the order of diffraction limit. Individual l-DNA molecules intercalated with the fluorescent dye YOYO-1 were investigated at subdiffraction spatial resolution by direct stochastic optical reconstruction microscopy（d STORM）. 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[17_TD$IF], 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.

Super-resolution morphological dissemination of intercalating dye in single DNA molecules via binding activated localization microscopy

Fluorescent dye （YOYO-I） intercalated with single DNA molecules were investigated via bindingactivated localization microscopy （BALM） at sub-diffraction limit resolutions. Various dye-to-DNA base pair （bp） ratios were imaged using the blinking property of YOYO-1 dye under optimum BALM switching buffer conditions. Individual DNA molecules exhibited regular/irregular intercalating phenomena with respect to dye-to-DNA bp ratio. The acquired images were reconstructed into super-resolution images by applying a Gaussian fit to the centroid of the point spread function. The YOYO-1 intercalated with λ-DNA possessed a non-homogeneous region due to the different binding modes of YOYO-1 with λ-DNA. Each binding mode was imaged at the sub-diffraction limit super-resolution. The distance between homogenously localized intercalating dyes within the DNA molecules was measured to be 34nm （n= 10; dye：DNAbp= 1：100） without photocleavage in 50mmol/L β-mercaptoethylamine buffer. The results were similar to those of the theoretical values without photocleavage in the base pairs of single DNA molecules below the diffraction limit. The results paved the way for an in-depth microscopic analysis of molecular variation with single λ-DNA molecules. With this method, it should be possible to analyze the exact base pair breakdown during various stages of cell apoptosis.