Method, computer program, and data processing unit for creating at least one correction value for correcting fluorescence intensities in a fluorescence image, and optical observation system

A computer-implemented method for creating at least one correction value for correcting fluorescence intensities in a fluorescence image obtained with an optical observation system, the optical observation system comprising an illumination system and an optical observation device in which the fluorescence image is formed by an image forming beam path, the method comprising:

• receiving or retrieving a parameter value of at least one parameter of the optical observation system which influences an observation of fluorescence intensity;
• and
• generating the at least one correction value for correcting the fluorescence intensities in the fluorescence image based on the received or retrieved parameter value and an influence of the at least one parameter of the optical observation system, which influences the observation of the fluorescence intensity, on the fluorescence intensities in the fluorescence image,
• wherein the at least one parameter of the optical observation system, which influences the observation of the fluorescence intensity, is a parameter of the illumination system,
• wherein at least one reference measurement with a reference concentration of a fluorescent dye is carried out with a reference parameter value for the at least one parameter which influences the observation of the fluorescence intensity to obtain a reference value for the fluorescence intensity at the reference concentration of the fluorescent dye,
• wherein a simulation of an expected fluorescence intensity is carried out, within the scope of which a change in the fluorescence intensity in comparison with the reference intensity is determined for a deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value, and
• wherein a compensation factor is determined, with which it is possible to compensate a change in the fluorescence intensity in a digital image which is caused by the deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value.

A non-transitory computer-readable storage medium to store a computer program for creating at least one correction value for correcting fluorescence intensities in a fluorescence image obtained with an optical observation system, the optical observation system comprising an illumination system and an optical observation device in which the fluorescence image is formed by an image forming beam path, the computer program comprising instructions which, when executed on a computer, cause the computer to:

• receive or retrieve a parameter value of at least one parameter of the optical observation system which influences an observation of fluorescence intensity;
• and
• generate the at least one correction value for correcting the fluorescence intensities in the fluorescence image based on the received or retrieved parameter value and an influence of the at least one parameter of the optical observation system, which influences the observation of the fluorescence intensity, on the fluorescence intensities in the fluorescence image,
• wherein the at least one parameter of the optical observation system, which influences the observation of the fluorescence intensity, is a parameter of the illumination system,
• wherein at least one reference measurement with a reference concentration of a fluorescent dye is carried out with a reference parameter value for the at least one parameter which influences the observation of the fluorescence intensity to obtain a reference value for the fluorescence intensity at the reference concentration of the fluorescent dye,
• wherein a simulation of an expected fluorescence intensity is carried out, within the scope of which a change in the fluorescence intensity in comparison with the reference intensity is determined for a deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value, and
• wherein a compensation factor is determined, with which it is possible to compensate a change in the fluorescence intensity in a digital image which is caused by the deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value.

A data processing unit for creating at least one correction value for correcting fluorescence intensities in a fluorescence image obtained with an optical observation system, the optical observation system comprising an illumination system and an optical observation device in which the fluorescence image is formed by an image forming beam path, the data processing unit comprising:

• a memory in which a computer program is stored;
• and
• a processor configured to:
• receive or retrieve a parameter value of at least one parameter of the optical observation system, which influences an observation of fluorescence intensity;
• generate the at least one correction value for correcting the fluorescence intensities in the fluorescence image based on the received or retrieved parameter value and an influence of the at least one parameter of the observation system, which influences the observation of the fluorescence intensity, on the fluorescence intensities in the fluorescence image;
• and
• output the at least one correction value,
• wherein the at least one parameter of the optical observation system, which influences the observation of the fluorescence intensity, is a parameter of the illumination system,
• wherein at least one reference measurement with a reference concentration of a fluorescent dye is carried out with a reference parameter value for the at least one parameter which influences the observation of the fluorescence intensity to obtain a reference value for the fluorescence intensity at the reference concentration of the fluorescent dye,
• wherein a simulation of an expected fluorescence intensity is carried out, within the scope of which a change in the fluorescence intensity in comparison with the reference intensity is determined for a deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value, and
• wherein a compensation factor is determined, with which it is possible to compensate a change in the fluorescence intensity in a digital image which is caused by the deviation of the parameter value of the at least one parameter which influences the observation of the fluorescence intensity from the reference parameter value.