Localizing a singularized fluorophore molecule by continuously moving a focused light beam around the molecule

A method of determining a molecule position of a singularized fluorophore molecule in an object, the method comprising:

• providing a light beam including fluorescence excitation light and fluorescence influencing light, wherein the fluorescence influencing light is identical to the fluorescence excitation light or wherein the fluorescence influencing light is fluorescence inhibition light provided in addition to the fluorescence excitation light;
• shaping and focusing the light beam such as to form a light intensity distribution having a central intensity minimum of the fluorescence influencing light;
• continuously shifting the light intensity distribution with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position of the singularized fluorophore molecule;
• individually registering a plurality of individual photons of fluorescence light emitted by the singularized fluorophore molecule due to excitation by the fluorescence excitation light;
• recording an intensity minimum position of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered;
• and, in response to each individual photon of the plurality of individual photons registered,
• updating the estimated position around which the track extends on basis of the recorded intensity minimum position, and
• at least one of
• reducing extensions of the track around the estimated position, and
• increasing a fluorescence influencing effectiveness of the fluorescence influencing light.

A laser-scanning microscope for determining a molecule position of a singularized fluorophore molecule in an object, the laser-scanning microscope comprising:

• a light source configured to provide a light beam including fluorescence excitation light and fluorescence influencing light, wherein the fluorescence influencing light is identical to the fluorescence excitation light or provided in addition to the fluorescence excitation light;
• a beam shaper and an objective configured to shape and focus the light beam such as to form a light intensity distribution having a central intensity minimum of the fluorescence influencing light;
• a scanner configured to continuously shift the light intensity distribution with regard to the object such that the central intensity minimum is continuously moved along a track repeatedly extending around an estimated position of the singularized molecule;
• a detector configured to individually register a plurality of individual photons of fluorescence light emitted by the singularized molecule due to excitation by the fluorescence excitation light;
• and
• a controller configured to record an intensity minimum position of the central intensity minimum for the excitation of each individual photon of the plurality of individual photons registered;
• wherein the controller is further configured to, in response to each individual photon of the plurality of individual photons registered,
• update the estimated position around which the track extends on basis of the recorded intensity minimum position, and
• at least one of
• reduce extensions of the track around the estimated position, and
• increase a fluorescence influencing effectiveness of the fluorescence influencing light.