Method and apparatus for high-resolution localization of a single emitter in multiple spatial directions

A method for high-resolution determination of the location of an excitable emitter in three spatial directions in a sample by scanning the excitable emitter with a 3D excitation donut having a central local minimum,

• wherein in an axial localization step, the central local minimum is sequentially placed at two axial sample positions on a sampling pattern axis passing through an estimated location of the excitable emitter, the pair of sample positions enclosing the estimated location of the excitable emitter,
• wherein in the axial localization step emission emitted from the excitable emitter for each of the axial sample positions is measured and the measured value is assigned to the respective axial sample position,
• wherein in the axial localization step a new estimate of the axial location of the excitable emitter is determined from the measured values associated with the axial sample positions,
• wherein
• in a lateral localization step, the central local minimum is placed exclusively in a sampling pattern plane oriented perpendicular to the sampling pattern axis sequentially at at least three lateral sample positions arranged around a location of the excitable emitter estimated in one or more steps performed earlier,
• wherein in the lateral localization step emission emitted from the excitable emitter for each of the lateral sample positions is measured and the measured value is assigned to the respective lateral sample position,
• wherein in the lateral localization step a new estimate of the lateral location of the excitable emitter is determined from the measured values associated with the lateral sample positions.