Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponent ial bleaching regime, is employed to determine separately the excitation in tensity and the fluorescence detection efficiency distributions in the fiel d of view of a confocal fluorescence microscope. We demonstrate that once t hese distributions are known, it is possible to correct an image of a speci men for intensity Variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is ind icated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the f luorescence intensities in an image of a specimen in terms of the fluoresce nce quantum yield, the absorption cross-section and the concentration of th e fluorophore in the specimen.