(A) The oligopaints used by Fields et al. contained a chromosome barcode (orange), a 3 Mb barcode (blue), a 500 kb barcode (purple), a 42-base region (grey) that hybridizes to a region of interest on a specific chromosome, and a 3′ barcode (green). The first stage in the process (pale blue boxed region) involves the hybridization of the 42-base region in the oligopaint to its target (1); a bridging oligo then hybridizes to one of the barcodes on the oligopaint (2); a detection oligo with a fluorescent dye then hybridizes to the bridging oligo (3). It is also possible (yellow boxed region) for a second bridging oligo to hybridize to the 3′ barcode on the oligopaint and increase the number of genome regions that can be detected within a single nucleus. (B) This approach can be used to determine the location of different chromosomes within the nucleus by using bridging oligos that bind to the chromosome barcode on the oligopaints specific to each chromosome, along with detection oligos with different fluorescent dyes (top schematic: green for chromosome II; red for chromosome IV). Likewise, the location of different regions within the same chromosome can be determined by using bridging oligos that bind to either the 3 Mb or 500 kb barcodes on the oligopaint specific to one chromosome (bottom schematic). Here blue fluorescence from the 3′ barcode and red fluorescence from the 500 kb barcode combine to give purple fluorescence; blue fluorescence from the 3′ barcode and green fluorescence from the 3 Mb barcode combine to give orange fluorescence. The dotted line represents the region of the nucleus occupied by chromosome IV.