MulticolorFISH

Multicolor FISH, short for multicolor fluorescent in situ hybridization, is a cytogenetic technique that uses a set of differently labeled DNA probes to tag multiple chromosomes or chromosomal regions in distinct colors. This color-coding enables the simultaneous visualization of chromosome structure and rearrangements within a single specimen, typically on metaphase spreads or interphase nuclei.

The method relies on fluorescent dyes or fluorophores attached to DNA probes that are complementary to specific chromosomal targets. After hybridization and washing, samples are examined under fluorescence microscopy. Modern implementations often employ spectral imaging or linear unmixing to separate overlapping emission spectra, allowing each chromosome or region to be identified by a unique color combination. Variants include whole-chromosome paint probes (M-FISH or SKY) and region-specific probe sets (multicolor banding, MCB) that produce band-like color patterns along chromosomes.

Variants and related approaches have been developed to improve resolution and interpretability. SKY (spectral karyotyping) uses a complete set of chromosome-specific probes with distinct spectral signatures, while M-FISH generalizes this concept to many colors for easier detection of complex rearrangements. MCB uses region-targeted probes to generate subchromosomal color patterns that resemble traditional G-banding.

Applications of multicolor FISH include cancer cytogenetics, where it aids in identifying translocations, inversions, duplications, and complex karyotypes, as well as constitutional or prenatal diagnostics and research into genome organization. Limitations include the need for high-quality samples and specialized imaging equipment, potential interpretation complexity, and limited resolution for detecting very small rearrangements.