Attenuation correction for confocal laser scanning microscopy and its application in chromatography

The applicability of confocal laser scanning microscopy is limited, e.g. by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on beam modelling and on a differential form of the modified Beer–Lambert law. It turns out that the intensity decay can be modelled as a double convolution of the microscopic image with the intensities of the excitation semibeam and the emission beam. Under weak assumptions made for the intensities of the fluorescent radiation and the detected signal, formulas for the attenuation correction and the attenuation simulation are derived. The method traces back to that one published by Roerdink which is modified concerning a more realistic beam modelling, avoiding the so‐called weak attenuation expansion and considering fluorescence excitation throughout the light cone of the excitation beam. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.