Analyse der Chromosomenverteilung in menschlichen Fibroblasten mittels 3D-Vielfarben Fluoreszenz in situ Hybridisierung
b'In human diploid fibroblasts (HDFs) the cell nucleus is oval in shape, quite large in xy-diameter (10-20\\xb5m), but flat in z-direction (5\\xb5m). In these nuclei chromosome territories typically lie side by side or slightly above each other. The question whether these arrangements are ordered or variable has yielded conflicting answers. We hybridised an improved 7-fluorochrome MFISH probe set on 3D-preserved cell nuclei, fixed with buffered 4% paraformaldehyde. A LEICA wide field microscope with an 8-filter-wheel and an automated z-step motor was used for imaging of the 7 fluorochromes plus DAPI. Multicolor images from nuclei were taken as serial sections in z-direction. After deconvolution, a specifically developed Program goldFISH Saracoglu K. et al. 2001 was used to classify the images according to the labelling scheme. The classification algorithm corresponds to the procedure previously used for metaphase spreads, now adopted to 3-D studies of chromosome territory arrangements in the cell nucleus. The analysis of 30 G0-fibroblast nuclei and 30 prometaphase rosettes revealed a pronounced variability of chromosome territory neighbourhoods, as described by Allison D. C. et al. 1999, but in contrast to Nagele R. et al. 1995. However we noted a distinct radial order: small chromosomes were located close to the centre while large chromosomes were positioned towards the nuclear rim. This non-random radial positioning could also be observed in prometaphase rosettes'