Method for adjusting a fluorescence image intensity, and fluorescence microscope

A fluorescence microscope, which should be adjusted to match a reference fluorescence microscope, comprising:

• an objective for imaging an object through an imaging beam path,
• a radiation source for generating illumination radiation having at least two different wavelengths simultaneously,
• at least one optical element for input coupling the illumination radiation into the imaging beam path,
• a detector unit for detecting fluorescence from the object through the imaging beam path, and
• a control unit configured to collect and evaluate measurement data from a detector of the detector unit, and
• a memory device for storing calibration data for adjusting a fluorescence image intensity of the fluorescence microscope to that of a reference fluorescence microscope,
• wherein the control unit is configured to implement the following:
• checking whether a set system configuration of the fluorescence microscope matches an entry of calibration data in the memory device of the fluorescence microscope,
• generating an adjustment shading correction by a shading reference sample using the fluorescence microscope to be adjusted,
• acquiring an adjustment fluorescence image of a qualified system reference calibration sample using the detector of the fluorescence microscope to be adjusted, with the set system configuration and using the previously generated adjustment shading correction,
• determining a mean adjustment fluorescence image intensity FIUsed of fluorescence, which was generated in the qualified system reference calibration sample, in a fluorescence channel of the fluorescence microscope to be adjusted,
• calculating and storing a correction factor KFI for the fluorescence channel from a mean interim fluorescence image intensity FIinter from the memory device and the mean adjustment fluorescence image intensity FIUsed in accordance with
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• wherein for the fluorescence microscope to be adjusted, the correction factor KFI is configured to render each adjustable system parameter, which depends linearly on the fluorescence image intensity, for the adjustment of the fluorescence image intensity correctable,
• wherein
• the qualified system reference sample is in the form of a homogeneously fluorescent glass substrate with emission bands between 400 nm and 850 nm.