A calibration system, comprising: - a fluorescence microscope; and - a calibration slide comprising a substrate, and a pixel layout comprising a plurality of spaced apart metal nanostructures arranged on a surface of the substrate and at least one further layout provided on the surface of the calibration slide, which is selected from the group consisting of: a monolayer of colored microbeads; a resolution and distortion test target, a layer of inorganic phosphors; wherein the metal nanostructures are arranged to produce plasmon resonances that allow absorbing light at an excitation wavelength to produce photo-luminescence and/or fluorescence light for generating a fluorescence image; wherein the fluorescence microscope comprises: - a light source; - a light detector; wherein the light source and the light detector are arranged in an optical path; wherein in calibration, the light source is configured to provide light to be absorbed by the metal nanostructures of the calibration slide positioned in the optical path at an excitation wavelength to produce said photo-luminescence and/or fluorescence light; and wherein the light detector is configured to detect the produced photo-luminescence and/or fluorescence light for acquiring fluorescent image data of a fluorescence image as calibration test data for calibration purposes. - a fluorescence microscope; and - a calibration slide comprising a substrate, and a pixel layout comprising a plurality of spaced apart metal nanostructures arranged on a surface of the substrate and at least one further layout provided on the surface of the calibration slide, which is selected from the group consisting of: a monolayer of colored microbeads; a resolution and distortion test target, a layer of inorganic phosphors; wherein the metal nanostructures are arranged to produce plasmon resonances that allow absorbing light at an excitation wavelength to produce photo-luminescence and/or fluorescence light for generating a fluorescence image; wherein the fluorescence microscope comprises: - a light source; - a light detector; wherein the light source and the light detector are arranged in an optical path; wherein in calibration, the light source is configured to provide light to be absorbed by the metal nanostructures of the calibration slide positioned in the optical path at an excitation wavelength to produce said photo-luminescence and/or fluorescence light; and wherein the light detector is configured to detect the produced photo-luminescence and/or fluorescence light for acquiring fluorescent image data of a fluorescence image as calibration test data for calibration purposes. - a light source; - a light detector; wherein the light source and the light detector are arranged in an optical path; wherein in calibration, the light source is configured to provide light to be absorbed by the metal nanostructures of the calibration slide positioned in the optical path at an excitation wavelength to produce said photo-luminescence and/or fluorescence light; and wherein the light detector is configured to detect the produced photo-luminescence and/or fluorescence light for acquiring fluorescent image data of a fluorescence image as calibration test data for calibration purposes.