This study provides a three-dimensional (3D) analysis of differences between the 
3D morphology of active and inactive human X interphase chromosomes (Xa and Xi 
territories). Chromosome territories were painted in formaldehyde-fixed, 
three-dimensionally intact human diploid female amniotic fluid cell nuclei (46, 
XX) with X-specific whole chromosome compositive probes. The colocalization of a 
4,6-diamidino-2-phenylindole dihydrochloride-stained Barr body with one of the 
two painted X territories allowed the unequivocal discrimination of the inactive 
X from its active counterpart. Light optical serial sections were obtained with 
a confocal laser scanning microscope. 3D-reconstructed Xa territories revealed a 
flatter shape and exhibited a larger and more irregular surface when compared to 
the apparently smoother surface and rounder shape of Xi territories. The 
relationship between territory surface and volume was quantified by the 
determination of a dimensionless roundness factor (RF). RF and surface area 
measurements showed a highly significant difference between Xa and Xi 
territories (P < 0.001) in contrast to volume differences (P > 0.1). For 
comparison with an autosome of similar DNA content, chromosome 7 territories 
were additionally painted. The 3D morphology of the chromosome 7 territories was 
similar to the Xa territory but differed strongly from the Xi territory with 
respect to RF and surface area (P < 0.001).