Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores

Analyzing the first and higher-order moments of the diffraction spot of a 4Pi fluorescence detection scheme facilitates two-color, three-dimensional super-resolution microscopy with ~6 nm axial and ~8–23 nm lateral resolution in a layer ~650 nm thick. We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with