Depth Intensity Correction of Biofilm Volume Data from Confocal Laser Scanning Microscopes

Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, these are the decay of intensities with increasing depth by finite transparency of the media used, the effects of extinction of the examined objects, the bleaching of the fluorochrome and stray light at surfaces within the volume under research. Under certain assumptions the decay of intensities can be estimated and used for a partial depth intensity correction. This estimation of the approximated intensity decay function is described and its correction effect is outlined. Volume and local distribution parameters of bacterial cultures marked by fluorescent in situ hybridization (FISH) are measured. Measurements with different corrections are compared with measurements of original data.