A method to extract homogeneous regions in 3-D confocal microscopy enabling compensation for depth dependent light attenuation

Abstract

The problem considered is to compensate for the attenuation of light with depth when recording stacks of images with a confocal microscope, working in the epifluorescence mode. A basic assumption is that there are regions of the specimen that are homogeneous in the sense that the attenuation is constant within the region. A method is presented to trace such regions in the stack of recorded images. It is also shown how the attenuation factor of the traced region is determined. Its inverse is the correction factor to be used when compensating for the attenuation relevant to the region. Techniques have been implemented to perform the compensation automatically by changing the photomultiplier tube voltage during the recording, using computer control. Some results of using this technique are reported.

To trace a homogeneous region, a stack of 2-D histograms of adjacent images, a histostack, is used. It usually shows distinguishable features through which homogeneous regions can be traced. A homogeneous region may be extracted from the stack of recorded images and dealt with separately, set apart in a special stack. The residual stack of images, containing what is left over after a homogeneous region has been extracted, is also available, and can be used for further extractions of homogeneous regions. In this way the extraction procedure can be used for image segmentation in three dimensions.

A review is given of the fundamentals of the extraction process.