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In order to separate different proteins, liquid chromatography is often used. The sample is pumped through a column filled with microspheres. The velocity of the proteins are depending on their interaction with the microspheres. The proteins could be labelled with a fluorescent marker and the distribution of the protein within the sphere can be recorded using a CSLM. When collecting optical sections using a CSLM the detected intensity decreases the deeper in the specimen the section is collected. This is due to absorption, scattering and bleaching. For the special case of a single microsphere it is of interest to find out how this combined effect is distributed within the sphere for a certain distribution of the fluorescent stain. When this distribution is known the attenuation can be compensated for. In the simulation the distribution of the stain is supposed to be the result of a diffusion process and all attenuation is supposed to arise from absorption only. The attenuation for a certain volume element (voxel) is supposed to occur from absorption in the voxels above, within the cone formed by the focused excitation light beam. A basic assumption is that the attenuation within each voxel is a fraction of the fluorescent intensity within that same voxel. A simulation program has been written where the parameters of the diffusion process within the microsphere can be controlled. Also the parameters for the attenuation calculation can be set, e.g. the assumed fraction of fluorescent intensity that act as attenuation. 3D datasets can be generated for visualization. Also intensity profiles can be generated along a diameter of the simulated sphere in the depth direction, since the intensity distribution is circularly symmetric in the lateral directions. Some comparisons are made to real microspheres, and the parameters are adjusted for closest resemblance. This adjustment can be done manually but an implementation using non-linear fitting of data is also presented. The simulated diffusion constant, fraction of intensity acting as attenuation, and the maximum intensity are fitted to experimental data from vertical slices of microspheres.
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