Method, minflux microscope and computer program for localizing or tracking emitters

A method for localizing or tracking an emitter in a sample using a MINFLUX method, wherein the MINFLUX method is a localization or tracking method for individual emitters in which a light distribution of illumination light that excites light emissions from the emitter is generated at the focus in the sample, wherein the light distribution comprises a local minimum and in which the position of an individual emitter is determined by detecting light emissions from the emitter for different positions of the minimum or for different light distributions in a close range of the emitter,

• wherein the sample is illuminated in the close range of the emitter by an illumination light beam having an intensity distribution of illumination light that comprises a central minimum, wherein the illumination light is excitation light, and wherein the illumination light excites the emitter to emit light,
• wherein the intensity distribution is displaced during a measurement cycle by a beam scanner comprising at least one optical component displacing the illumination light beam to scanning positions,
• and wherein emissions from the emitter are detected for the scanning positions and assigned to the scanning positions,
• wherein the beam scanner is controlled by control signals to displace the intensity distribution along a continuous path during the measurement cycle, wherein the intensity distribution is displaced along the path both to scanning positions in an outer region of the close range of the emitter and to scanning positions in an inner region of the close range of the emitter, wherein the scanning positions are arranged around an expected position of the emitter in the sample in at least two spatial directions, and wherein the control signals and/or nominal movements of the at least one optical component can be described respectively as a function of time by a sine function or by a weighted sum of sine functions with a finite number of summands,
• wherein the sine function has a time-constant frequency and a time-constant amplitude over the measurement cycle or the sine functions of the sum have respective time-constant frequencies and respective time-constant amplitudes over the measurement cycle,
• and wherein a position of the emitter is estimated from a plurality of emissions from the emitter and a plurality of scanning positions assigned to the emissions.

A MINFLUX microscope for localizing or tracking an emitter in a sample according to a MINFLUX method, wherein the MINFLUX method is a localization or tracking method for individual emitters in which a light distribution of illumination light that excites light emissions from the emitter is generated at the focus in the sample, wherein the light distribution comprises a local minimum and in which the position of an individual emitter is determined by detecting light emissions from the emitter for different positions of the minimum or for different light distributions in a close range of the emitter, wherein the MINFLUX microscope comprises

• a light source configured to generate an illumination light beam,
• an illumination optic configured to illuminate a sample in a close range of an emitter by an intensity distribution of illumination light that comprises a central minimum, wherein the illumination light is excitation light or emission inhibition light, in particular STED light, and wherein the illumination light or another light excites the emitter to emit light,
• a beam scanner comprising at least one optical component that displaces the illumination light beam, wherein the beam scanner is configured to displace the intensity distribution to scanning positions during a measurement cycle, and
• a measuring device configured to detect emissions from the emitter for the scanning positions,
• a control unit which is configured to control the beam scanner with control signals to displace the intensity distribution along a continuous path during the measurement cycle, so that the intensity distribution is displaced along the path both to scanning positions in an outer region of the close range of the emitter and to scanning positions in an inner region of the close range of the emitter, wherein the scanning positions are arranged around an expected position of the emitter in the sample in at least two spatial directions, and wherein the control signals and/or nominal movements of the at least one optical component can be described respectively as a function of time by a sine function or by a weighted sum of sine functions with a finite number of summands, wherein the sine function has or the sine functions of the sum have a time-constant frequency and a time-constant amplitude over the measurement cycle, and
• a computing unit configured to estimate a position of the emitter from a plurality of emissions from the emitter detected by the measuring device and a plurality of scanning positions assigned to the emissions.