Many studies have suggested a link between the spatial organization of genomes 
and fundamental biological processes such as genome reprogramming, gene 
expression, and differentiation. Multicolor fluorescence in situ hybridization 
on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal 
microscopy, has become an effective technique for analyzing 3D genome structure 
and spatial patterns of defined nucleus targets including entire chromosome 
territories and single gene loci. This technique usually requires the 
simultaneous visualization of numerous targets labeled with different colored 
fluorochromes. Thus, the number of channels and lasers must be sufficient for 
the commonly used labeling scheme of 3D-FISH, "one probe-one target". However, 
these channels and lasers are usually restricted by a given microscope system. 
This paper presents a method for simultaneously delineating multiple targets in 
3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other 
labeling schemes, this method is convenient and simple for multicolor 3D-FISH 
studies, which may result in widespread adoption of the technique. Lastly, as an 
application of the method, the nucleus locations of chromosome territory 18/21 
and centromere 18/21/13 in normal human lymphocytes were analyzed, which might 
present evidence of a radial higher order chromatin arrangement.