Fluorescence in situ hybridization (FISH) is a kind of cytogenetic technique which uses fluorescent probes binding parts of the chromosome to show a high degree of sequence complementarity, and it  can be used to find out where the fluorescent probe bound to the chromosome. FISH is often used for finding specific features in DNA, and to detect and localize specific RNA targets (mRNA, lncRNA and miRNA).This technique provides a novel way for researchers to visualize and map the genetic material in an individual cell, including specific genes or portions of genes. Different from most other techniques used for chromosomes study, FISH has no need to be performed on cells that are actively dividing, which makes it a very versatile procedure. It is composed by 4 main passages:

·         A probe complementary to the known sequence is made and it is labelled with a fluorescent marker, as for example fluorescein, by incorporating nucleotides that have the marker attached to them;

·         Chromosomes are put on a microscope slide and denatured;

·         The probe is denatured and added to the microscope slide, allowing the probe hybridize to its complementary site;

·         The excess probe is washed off and the chromosomes are observed under a fluorescent microscope. The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to.

FISH is widely used for several diagnostic applications as for example identification of numerical and structural abnormalities, characterization of marker chromosomes, monitoring the effects of therapy, detection of minimal residual disease. Moreover it has many applications in research as gene mapping or identification of amplified genes. FISH is also used to compare the genomes of two biological species to deduce evolutionary relationships.

A techniqe which derives from FISh is  the multiplex in situ hybridization (M-FISH). It represents one of the most significant developments in molecular cytogenetics of the past decade; it is a 24-color karyotyping technique and is the method of choice for studying complex interchromosomal rearrangements. The process is composed by three main steps:

·         The multiplex labeling of all chromosomes in the genome with finite numbers of spectrally distinct fluorophores such that each homologous pair of chromosomes is uniquely labelled;

·         The microscopic visualization and digital acquisition of each fluorophore using specific single band-pass filter sets and dedicated M-FISH software. These acquired images are then superimposed enabling individual chromosomes to be classified based on the fluor composition in accordance with the combinatorial labeling scheme of the M-FISH probe cocktail used;