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Localization microscopy relies on the separation of the emission of individual fluorophores within a diffraction limited spot. In popular techniques like PALM, STORM, or blinking the structure of interest is first labeled and lasers/chemicals are then adjusted such that only a sparse subset of all labels is fluorescent at the same time. We propose an alternative strategy, namely to use fluorophores that are “switched on” when bound to the target structure and to localize them while they are binding in the presence of free dye. This approach is best described by the term Binding-Activated Localization Microscopy (BALM). Here we demonstrate BALM with DNA-binding dyes that show a strong fluorescence enhancement when bound to dsDNA. Surface-immobilized DNA molecules were imaged with a resolution of∼ 14 nm (full-width at half-maximum), reaching a spatial sampling of nearly 1/nm. We further show …