Microscopy system and microscopy method for quantifying a fluorescence

A microscopy method for quantifying a fluorescence of protoporphyrin IX, the method comprising:

• imaging an object region onto a first detector field having a multiplicity of first pixels,
• wherein at least one first optical filter with a first wavelength-dependent transmission characteristic is arranged in a first beam path between the object region and each one of the pixels of the first detector field;
• imaging the object region onto a second detector field having a multiplicity of second pixels, wherein at least one second optical filter with a second wavelength-dependent transmission characteristic that differs from the first wavelength-dependent transmission characteristic is arranged in a second beam path between the object region and each one of the pixels of the second detector field;
• exciting at least a first fluorescence and a second fluorescence in the object region, wherein the first fluorescence is the fluorescence of protoporphyrin IX;
• recording a first image of the imaged object region using the first detector field;
• recording a second image of the imaged object region using the second detector field;
• and
• determining a spatially dependent fluorescence intensity of the first fluorescence in the object region by determining in each case a fluorescence intensity value for a plurality of first pixels or a plurality of groups of first pixels in the first detector field, the fluorescence intensity value representing a fluorescence intensity at a location in the object region imaged onto the respective first pixel or the respective group of first pixels,
• wherein the fluorescence intensity value is determined from a radiation intensity detected by the respective first pixel or group of first pixels of the first detector field, a wavelength-dependent detection efficiency of the respective first pixel or group of first pixels, a radiation intensity detected by a second pixel or group of second pixels of the second detector field on which the location in the object region is imaged, a wavelength-dependent detection efficiency of the second pixel or group of second pixels, a fluorescence spectrum of the first fluorescence, and a fluorescence spectrum of the second fluorescence, and
• wherein, for a first wavelength and a second wavelength:
• a transmission at the first wavelength of the at least one first filter arranged between a location (x,y) in the object region and the first pixel (i,j) or group (i,j) of first pixels of the first detector field on which the location (x,y) is imaged is at least 1.5 times greater than the transmission at the first wavelength of the at least one second filter arranged between the location (x,y) in the object region and the second pixel (k,l) or group (k,l) of second pixels of the second detector field on which the location (x,y) is imaged, and
• the transmission at the second wavelength of the at least one first filter arranged between the location (x,y) in the object region and the first pixel (i,j) or group (i,j) of first pixels of the first detector field is at least 1.5 times smaller than the transmission at the second wavelength of the at least one second filter arranged between the location (x,y) in the object region and the second pixel (k,l) or group (k,l) of second pixels of the second detector field.