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

Fluorescent dye (YOYO-1) intercalated with single DNA molecules were investigated via binding-activated 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 34 nm (n = 10; dye:DNAbp = 1:100) without photocleavage in 50 mmol/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.