A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 Fm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-BOO (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.Key terms: DNA cytometry, confocal laser microscopy, histology, 3-D There is a considerable interest in DNA cytometry, notably because of the importance of searching for correlations of nuclear DNA content with cancer prognosis in individual patients (e.g., 22, 40, 62).The two methods commonly used a t present for nuclear DNA content analysis, image cytometry (ICM) and flow cytometry (FCM), are not devoid of limitations. Observing the tissue architecture may be important in identifying regions for cytometry and, therefore, the main shortcoming of ICM on smears or imprints and of FCM is obviously that they do not allow the study of DNA contents in situ. Even ICM on histological sections does not allow this, as the tissue structure is observed in projection and the resulting nuclear profiles are usually either incomplete if the section is thin or overlapping if the section is thick enough to include a sufficient proportion of complete nuclei. Finally, preparation procedures for ICM and FCM may produce a selection bias of a given cell type (9) and/or nuclear debris (27).'This paper is an expanded version of a work presented at the

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SUMMARY Confocal scanning laser microscopy has been used to make three‐dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno‐labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double‐fluorescence emission requires an image processing step for the correction of ‘contamination’ effects due to the overlap between fluorescein and Texas Red emission spectra. At the pre‐natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells. Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18–20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two‐cell‐thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one‐cell‐thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.

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