A versatile image analysis approach for simultaneous chromosome identification and localization of FISH probes

Modern cytogenetic techniques, such as comparative genomic hybridization (CGH) and the multi-color fluorescence in situ hybridization (FISH) techniques of multiplex fluorescence in situ hybridization (M-FISH) and spectral karyotyping (SKY), require a coordinated banding analysis to maximize their usefulness. All of the methods currently used, including Giemsa (G-) banding, Alu banding, and 4',6-diamidino-2-phenyl-indole (DAPI) banding, have serious drawbacks. A simple and effective method to band chromosomes concurrently with FISH is needed. To address this problem, we stained chromosomes with DAPI and chromomycin A3, and then used an image analysis program to generate banding by dividing the image taken with a DAPI excitation filter by the image taken with a chromomycin A3 excitation filter. The result was a metaphase spread in which the chromosomes possessed a banding pattern characteristic of R-banding. The image analysis program was then used to generate linescans of pixel intensity versus relative position along the length of chromosomes that were banded using this technique, which we have called D/C R-banding. Each chromosome in a genome was represented by a characteristic scan profile, which was unaffected by FISH signals. Reference linescans were prepared by karyotyping D/C R-banded chromosomes for a given species, and then drawing lines along the length of the known chromosomes. The linescans were combined into a spreadsheet database, which was linked by dynamic data exchange to the image analysis program and normalized for length and intensity. The linescan of an unknown chromosome was then transferred to the spreadsheet, where it was normalized for length and intensity and overlaid on the linescans of each chromosome in the genome. Unknown chromosomes were identified by comparison of their graphs with graphs in the standardized reference genome. We have used this approach to create reference linescan karyotypes of several species, and to identify chromosomes on which FISH was performed.