Fluorescence photobleaching-based shading correction for fluorescence microscopy

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A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.

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