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SUMMARY This study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomal in situ suppression hybridization with chromosome‐specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three‐dimensional (3‐D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were extracted by applying the Delaunay graph, the dual of the Voronoi diagram, which describes the neighbourhood in the Voronoi diagram. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3‐D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X‐ and 7‐chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X‐territories but not between chromosome 7‐territories.

A tilting device for biological specimens (rotation angle up to 27r), especially fluorescence-labelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adapter for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser fluorescence microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor.The three-dimensional (3-D) image-forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that in spite of aberration effects, the optical arrangement used allows a gain in the 3-D resolution by tilting the object.A low-cost, fully automated 3-D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black-and-white CCD camera. The system was operated by a personal computer. The online visualization ('movie') of rotating objects indicates the feasibility of the system.

Chromosome territories 5, 7 and X were painted in female human amniotic fluid or fibroblast cell nuclei with chromosome specific DNA library probes. For probe detection the fluorochromes FITC or TRITC were applied. Using confocal laser scanning fluorescence microscopy ten to twenty light optical sections of FITC or TRITC images were registered with equal spacing (approximately 400 nm) for each of 131 nuclei showing well separated homologous chromosome territories. Chromosome territory areas were segmented applying a range of gray value thresholds to each section. For each gray value threshold the territory volumes for a given pair of homologs were computed by means of the Cavalieri estimator. The larger volume was divided by the smaller one to obtain a volume ratio. Between 9 and 26 territory volume ratios were calculated for a pair of homologs in a given nucleus. This approach yielded the following results: (i) the range of volume ratios was very similar for both autosome and X‐chromosome territories and (ii) most volume ratios (> 70%) were between 1 and 1.5. The same results were obtained for cells fixed with 4% buffered formaldehyde or methanol/acetic acid indicating that volume ratio measurements are relatively robust against shrinkage effects induced by the latter type of fixation. These similarities of volume ratios argue against the view that the genetic inactivation of one X‐chromosome territory in female cell nuclei should be correlated with a decrease of its volume. Such a decrease was expected in case of a strong overall condensation of this chromosome as compared to other chromosomes.

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