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 colours 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.