Three-dimensional image processing method to compensate for depth-dependent light attenuation in images from a confocal microscope

When looking into the depth of a semitransparent specimen, using a confocal laser microscope working in the epifluorescence mode, it is often observed that the recorded images are darker the deeper the optical sections are located in the specimen. One reason for this is that light is absorbed in the specimen on its way to and from the section. A manual method to compensate for this darkening is to vary the electronic amplification at the recording. The appropriate amplification depends not only on the depth but also on the specimen, its shape and density, etc. Methods to replace the manual adjustments with computer methods, applied to stacks of uncompensated images recorded at different equidistant depths, have been suggested. A basic assumption is then that there are regions of the specimen that are homogeneous enough to serve as reference regions for the compensation. A key problem is to detect these regions. An interactive method to trace homogeneous regions in a stack of recorded images is described. It is also shown how image segmentation can be performed to extract such regions.