Analysis of the spatial distribution of AgNOR proteins in cell nuclei using simultaneous confocal scanning laser fluorescence and transmitted light microscopy

The selective staining of nucleolar acidic proteins by silver dyes (AgNOR technique) is a promising tool to discriminate malignant cells from benign cells in tumour pathology. Until now, the existing methods to measure the distribution of the AgNOR proteins were performed in two-dimensional space. Confocal scanning laser microscopy (CSLM) and progress in computer sciences make it possible to image biological specimens in three dimensions. We have used these techniques to develop a method to discriminate cells by comparing the three-dimensional (3-D) spatial distribution of the AgNOR proteins. The distribution of the AgNOR proteins was described by the following measurements: the volume fraction of the nucleus occupied by the AgNOR-stained aggregates, the distances between the aggregates, their number per nucleus, the distance of each aggregate from the nuclear border and their anisotropy. CSLM was used to acquire simultaneously the 3-D data set of the nucleus (confocal fluorescence) and the 3-D data set of the location of the AgNOR proteins (brightfield transmission). Two biases, due to the different modes of microscopy and the silver labelling technique, had to be taken into account in the evaluation of the measurements. A preliminary study on three lines of lymphocytic cells shows that these parameters are discriminatory. Thus, this method makes it possible to combine two different modes of scanning laser microscopy for a 3-D quantitative analysis and is potentially useful for assessing the spatial distribution of AgNOR proteins in tumour pathology.