Karyotype   analysis   by   means   of    chromosome banding techniques   is   the   pillar   of   cytogenetic diagnostics, both in the clinical and  in   the   cancer cytogenetic laboratory. Using chromosome banding alone,   however, a   disturbingly   high   number   of chromosomal   aberrations cannot be characterized comprehensively. We   have   therefore developed a novel      karyotyping   approach,   termed    spectral karyotyping,     that   is    based on the simultaneous hybridization of 24   combinatorially  labelled human chromosome painting probes. The   visualization  of all human    chromosomes   in   different   colors    is achieved by   spectral   imaging.   Spectral   imaging combines fluorescence  microscopy,   CCD-imaging and    Fourier        spectroscopy        to       visualize, simultaneously,   the  entire    spectrum  at all image points. Here we describe the  principle   of   spectral imaging,        define         software     and    hardware requirements, and present relevant  applications   of the technique to cytogenetics and cytology.