Spatially modulated illumination microscopy allows axial distance resolution in the nanometer range.

For an improved understanding of the structural basis of cellular mechanisms, it is highly desirable to develop methods for a detailed topological analysis of biological nanostructures and their dynamics in the interior of three-dimensionally conserved cells. We present a method of far-field laser fluorescence microscopy to measure relative axial positions of pointlike fluorescent targets and the distance between each target in the range of a few nanometers. The physical principle behind this approach can be extended to the determination of three-dimensional (3D) positions and 3D distances between any number of objects that can be discriminated owing to their spectral signature, thus allowing topological measurements so far regarded to be beyond the capabilities of light microscopy.