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3-D image analysis, centromeric probes, chromosome 15, chromosome 7, confocal microscopy, FISH, nuclear architecture
Abstract:
The three-dimensional positions of two centromeric DNA probes specific for chromosomes 7 and 15 were determined using optical sectioning by multi-wavelength confocal laser scanning microscopy of human interphase fibroblasts. In addition to the fluorescence in situ hybridization (FISH) signals, the immunofluorescence of nuclear lamin was used to delineate the nuclear boundary. The coordinates of the in situ probes and the centre of the nucleus were determined for 38 nuclei from which the vectors and distances between homologous and heterologous centromeres and from each centromere to the nuclear centre were computed. The distributions of the calculated distances were analysed statistically and compared with those predicted for a random distribution. It was found that both chromosome loci are non-randomly distributed in the nucleus and that centromere 15, compared with centromere 7, is significantly closer to the centre of the nucleus. We established that quantitative three-dimensional (3-D) measurements of multiple targets are feasible although the experimentally and computationally intensive methods limit the attainable dataset to a relatively small number of nuclei. Therefore, the results were compared with those acquired for a larger number of similar cells in a simpler two-dimensional (2-D) analysis carried out in a companion study.
