AbstractIn the preparation of fluorescencein situhybridization (FISH) specimens, multiple probes, each tagged with a different fluorophore, are often used in combination. This allows several different molecular components to be visualized simultaneously. Usually their relative positions within the specimen are of scientific interest. Multi‐band fluorescence filter sets permit one to view multi‐labelled specimens in color.Given the emission spectra of available fluorophores, and the sensitivity spectra of available cameras and filter sets, it is impossible to obtain complete isolation of each fluorophore to a separate color channel. Even with a monochrome camera and carefully selected fluorophores and filters, spectral overlap still spreads each fluorophore across the color channels.This paper presents a processing method that effectively removes the spreading of fluorophore brightness among the color channels, thereby isolating each fluorophore to a single monochrome image. This simplifies subsequent image analysis, since each molecular component of the specimen can be analysed independently, but in register with the other components. Displayed images also are easier to interpret, since the colors appear more satura