Molecular-scale axial localization by repetitive optical selective exposure

We introduce an axial localization with repetitive optical selective exposure (ROSE-Z) method for super-resolution imaging. By using an asymmetric optical scheme to generate interference fringes, a <2 nm axial localization precision was achieved with only ~3,000 photons, which is an approximately sixfold improvement compared to previous astigmatism methods. Nanoscale three-dimensional and two-color imaging was demonstrated, illustrating how this method achieves superior performance and facilitates the investigation of cellular nanostructures. ROSE-Z achieves axial interference through an asymmetrical optical scheme, yielding 2 nm axial localization precision with ~3,000 photons and a single objective, which offers improved multicolor three-dimensional localization microscopy for cellular structures.