Restoration of confocal images for quantitative image analysis

SUMMARY Quantitative studies of three‐dimensional (3‐D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self‐absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction‐induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z‐resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3‐D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluorescence images of biological objects, such as interphase cell nucei.