The multiplicity of a FISH procedure is defined as the number of DNA targets that can be distinguished on the basis of optical properties, usually fluorescence color. In the simplest application of multiplicity, different fluoro-chromes that are spectrally well separated are attached to separate probes either directly or indirectly. For the visible part of the electromagnetic spectrum, blue, green, and red fluorescent dyes are available, permitting a multiplicity of 3 for visual observation of FISH results (Nederlof et al., 1989). When imaging devices sensitive to infrared light rays are used, for example a CCD camera with light integration capabilities, multiplicity can be increased to 4 or 5. When the targets are spatially separated, as in well-spread metaphase chromosomes, multiplicity can be increased by combinatorial labeling of the targets (Nederlof et al., 1990; Ried et al., 1992a; Wiegant et al., 1993; unit 8.3). Here multiplicity is 2n - 1, where n is the number of spectrally resolvable fluoro-chromes; this implies that with 3 and 4 fluoro-chromes, multiplicities of 7 and 15, respectively, can be achieved. For such combinatorial labeling of multiple FISH targets the ratios of the fluorescence intensities of the probes are in principle not relevant, but because the fluorescence intensity ratios of differentially labeled probes recognizing the same target turn out to be fairly constant after FISH (Nederlof et al., 1992), multiplicity can easily be increased to 12 (Dauwerse et al., 1992). Recently, successful FISH imaging of all the human chromosomes in 24 colors, using combinatorial FISH with five fluorochromes and either small band excitation/emission filter sets or special spectral imaging devices, has been reported (Schröck et al., 1996; Speicher et al., 1996).

Detection efficiency is an important issue in multicolor FISH, and it is generally advisable to apply high-multiplicity FISH only to large targets. For example, with FISH detection efficiencies are 0.9 and an attempted multiplicity of 6, only in 0.96 x 100, or 53%, of the cells will all six targets be visible simultaneously.

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