Validation of image processing tools for 3-D fluorescence microscopy

3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.