Method for high-resolution determination of the position of an excitable fluorophore in three spatial directions in a sample by scanning the excitable fluorophore with a 3D excitation donut having a central local minimum , - wherein, in an axial localisation step, the central local minimum is sequentially placed at two axial sample positions on a scan pattern axis passing through an estimated location of the excitable fluorophore , the pair of sample positions enclosing the estimated location of the excitable fluorophore , - wherein in the axial localisation step, fluorescence emitted by the excitable fluorophore is measured at each of the axial sample positions and the measured value is assigned to the respective axial sample position , - wherein in the axial localisation step, a new estimate of the axial position of the excitable fluorophore is determined from the measured values assigned to the axial sample positions , characterised in that - in a lateral localisation step following the axial localisation step, the central local minimum is placed exclusively in a scan pattern plane oriented perpendicular to the scan pattern axis sequentially at at least three lateral sample positions arranged around a estimated position of the excitable fluorophore estimated in one or more steps performed earlier, - wherein in the lateral localisation step, fluorescence emitted by the excitable fluorophore is measured at each of the lateral sample positions and the measured value is assigned to the respective lateral sample position , - wherein in the lateral localisation step, a new estimate of the lateral position of the excitable fluorophore is determined from the measured values assigned to the lateral sample positions . - wherein, in an axial localisation step, the central local minimum is sequentially placed at two axial sample positions on a scan pattern axis passing through an estimated location of the excitable fluorophore , the pair of sample positions enclosing the estimated location of the excitable fluorophore , - wherein in the axial localisation step, fluorescence emitted by the excitable fluorophore is measured at each of the axial sample positions and the measured value is assigned to the respective axial sample position , - wherein in the axial localisation step, a new estimate of the axial position of the excitable fluorophore is determined from the measured values assigned to the axial sample positions , characterised in that - in a lateral localisation step following the axial localisation step, the central local minimum is placed exclusively in a scan pattern plane oriented perpendicular to the scan pattern axis sequentially at at least three lateral sample positions arranged around a estimated position of the excitable fluorophore estimated in one or more steps performed earlier, - wherein in the lateral localisation step, fluorescence emitted by the excitable fluorophore is measured at each of the lateral sample positions and the measured value is assigned to the respective lateral sample position , - wherein in the lateral localisation step, a new estimate of the lateral position of the excitable fluorophore is determined from the measured values assigned to the lateral sample positions . <EOS>