Short-cut method for the correction of light attenuation influences in the experimental data obtained from confocal laser scanning microscopy.

Based on Lambert-Beer law and the light attenuation model, a new method will be introduced in this work in order to eliminate the disturbances caused by signal attenuation in the experimental data measured by confocal laser scanning microscopy (CLSM). This new method considers the attenuation effects which depend on concentration of fluorophore-labelled protein as well as attenuation effects which are independent from protein concentration. Furthermore, no solvent additive is required in order to match the refraction index of solvent to bead material. The determination of correction factors is, thus, easily done using the currently investigated chromatographic phase system, so that the validity of the correction factors in the current system can be guaranteed. The introduced correction method has been applied for the investigation of intraparticle protein distribution inside an HIC (hydrophobic interaction chromatography) particle.