Method and microscope for determining a fluorescence intensity

A method for evaluating a two-dimensional microscope image containing intensity distributions of fluorescence events of a sample marked with at least one fluorescent dye, wherein the intensity distributions have a respective diffraction-related extent, which corresponds to an extent of a point spread function of the microscope, and at least one intensity distribution of an individual one of the fluorescence events is isolated, comprising the following steps:

• ascertaining an integrated first intensity value in a first partial region of an overlap-free intensity distribution, in particular in the entire isolated intensity distribution;
• ascertaining an integrated second intensity value in a second partial region of an overlap-free intensity distribution, wherein the second partial region differs from the first partial region, in particular is smaller than the first partial region and is contained therein;
• and
• ascertaining a photometric characteristic variable of the isolated intensity distribution on the basis of the first intensity value and of the second intensity value, in particular on the basis of a quotient of the two intensity values;
• wherein the isolated intensity distribution is assigned a position in a third spatial dimension on the basis of the photometric characteristic variable, in particular with storage of the microscope image together with the assigned position.

A method for ascertaining a fluorescence intensity, comprising:

• a) recording a plurality of two-dimensional individual images of a sample using a microscope, wherein the same sample region is contained in each of the individual images and different emitters are excited to emit fluorescence during the recording of the individual images such that, in at least one of the individual images, at least one diffraction-limited intensity distribution of a fluorescence emission of an emitter is isolated;
• b) ascertaining the individual image with the isolated intensity distribution;
• c) ascertaining a first intensity value in a partial region of the isolated intensity distribution, in particular in the entire isolated intensity distribution, by integrating the recorded intensities of relevant image elements of the individual image;
• d) ascertaining at least one other of the individual images, which is, at least in an image region corresponding to the partial region of the isolated intensity distribution, free from fluorescence emissions;
• e) ascertaining a second intensity value in the image region of the other individual image by integrating the recorded intensities of relevant image elements of the other individual image;
• and
• f) ascertaining an adjusted fluorescence intensity as a difference between the first intensity value and the second intensity value;
• g) ascertaining a second adjusted fluorescence intensity for a second partial region of the isolated intensity distribution on the basis of steps a) to f), wherein the second partial region is different from the first partial region, in particular smaller than it and in particular is contained in it, and for ascertaining the second intensity value either i) the recorded intensities of the image elements of the relevant image region in the individual image or in the other individual images are integrated, or ii) the second intensity value for the first partial region is scaled to the area of the second partial region;
• and
• h) ascertaining a coordinate value for the emitter on the basis of the first adjusted fluorescence intensity and the second adjusted fluorescence intensity, in particular on the basis of a quotient of the first adjusted fluorescence intensity and the second adjusted fluorescence intensity.