The combination of multiple dye-DNA interactions, a fluorescence digital imaging system with a scientific CCD camera, and multivariate image analysis allows the rapid karyotyping of fluorescent human metaphase chromosome spreads. Chromosomes are stained with the bisbenzimidazole dye Hoechst 33342 and chromomycin A,, a dye pair used frequently in bivariate flow analysis and sorting of metaphase chromosomes in suspension. The use of ratio functions involving the total and peak intensities of the two dyes provides increased resolution of the karyotype in the microscope, and it can be anticipated that the same approach could lead to improved performance with flow systems as well. High pass filtering with a Laplace operator yields characteristic banded images of the individual chromosomes, even with total fields that are less than 200 pixels on a side.Key terms: CCD camera, bis-benzimidazole, chromomycin, cytogenetics Sam Latt's career exemplified the productive interface between medical problems (in particular, human genetics) and biophysical techniques. The application of fluorochromes in staining metaphase chromosomes by Caspersson and coworkers (9) revolutionized karyotype identification and, thereby, the field of human genetics. Latt identified energy transfer and quenching as the predominant determinants of chromosome banding patterns (4,6,22,29), thus providing a framework by which existing data on chromosome karyotyping could be evaluated. He introduced the use of the bisbenzimidazole dyes for chromosome staining and investigated their physico-chemical properties on normal and bromodeoxyuridine 25,26). His papers on increased contrast in chromosome banding due to dye pair-DNA interactions (19,21,23,24,29) assisted in the evaluation of various genetically inherited translocations.The original studies of Caspersson and coworkers demonstrating that most human chromosomes have a unique DNA content were the basis of the first quantitative cytochemical determinations (7) as well as of existing classification schemes based on UV absorption (27). The extension of karyotyping to flow cytometry utilized fluorochrome staining protocols which had proven effective in the quantitation of cellular DNA (2,11,13,17). Initial univariant flow karyotypes (8,321 with insufficient resolution were supplanted by bivariant flow karyotypes (14,181 based on the dye interaction studies of Latt and coworkers and the implementation of dual laser beam flow cytometers (31). Although all of the human chromosomes except 9-12 and 14 and 15 can generally be distinguished by flow karyotyping, detection of clinically significant chromosomal abnormalities has been hindered by problems related to the preparation of high-quality chromosome suspensions from small numbers of cells and of homolog variations between individuals. Slit-scanning flow cytometry can provide additional resolution (5,10, and references therein), but it is still imperative to perform morphological observations with a microscope in order to substantiate the presence and nature o...
