RESTORATION OF CONFOCAL IMAGES FOR QUANTITATIVE IMAGE-ANALYSIS

Quantitative studies of three-dimensional (3-D) structure of microscop ic objects have been made possible through the introduction of microsc opic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its speci fic imaging properties give rise to distortions in the images and hamp er subsequent quantitative analysis. Therefore, it is a prerequisite t hat confocal images are restored prior to analysis. The distortions ca n be divided into several categories: attenuation of areas in the imag e due to self-absorption, bleaching effects, geometrical effects and d istortions due to diffraction effects. Of these, absorption and diffra ction effects are the most important. This paper describes a method ai med at the correction of diffraction-induced distortions. All the step s necessary in restoring confocal images are discussed, including a no vel method to measure instrumental properties on a routine basis. To t est the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z-res olution and no ringing artefacts. The relevance of the method for imag e analysis is demonstrated by a comparison of results of applying 3-D texture analysis to restored and unrestored images of a synthetic obje ct. Furthermore, the method can be successfully applied to noisy fluor escence images of biological objects, such as interphase cell nucei.