Fluorescence Microscopy: Multicolor Fluorescence in situ Hybridization (FISH) Explained
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Fluorescence microscopy has become an essential tool in biomedical sciences since Sir George G. Stokes first described fluorescence in 1852. It took fluorescence in situ hybridization more than 100 years to follow. But in the last 15 years, a revolution in light microscopy fluorescence techniques has resulted in the unprecedented locating of, identifying, and recording data on the genetic makeup of biomedical samples.
What Is Fluorescence in situ Hybridization?
Fluorescence in situ hybridization (FISH) is a technique that uses fluorescent probes that bind only to the parts of a nucleic acid sequence with a high degree of sequence complementarity. A probe is a single strand of DNA or RNA that is complementary to a nucleotide sequence of interest. Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use with the Human Genome Project.
FISH was originally developed by biomedical researchers to localize the presence or absence of DNA sequences or chromosomes. Fluorescence microscopy is then used to find out where the probe is bound to the chromosomes.
If the fluorescent signal is weak, it must be amplified so that it can be detected by the fluorescence microscope. Fluorescent signal strength depends on probe labeling efficiency, the type of probe, and the type of dye.
Chromosomes must be firmly attached to a substrate, which is usually glass. Wash steps remove the unhybridized and partially hybridized probes. The results are then visualized and quantified using a fluorescence microscope that is able to excite the dye and record the image.
Where Is Fluorescence in situ Hybridization used?
FISH is used to find specific features in DNA. Some medical applications include genetic counseling, medicine, and species identification. FISH is also used to find specific RNA targets in cells, tumor cells, and tissue samples. FISH can help define the spatial-temporal patterns of gene expression in cells and tissues.
When parents have a child with a developmental disability, they often want to know more about the condition. FISH can be used to analyze the parents’ and child’s DNA. Even if a child’s disability is not understood, its cause may be able to be determined by using FISH and cytogenetic techniques.
Some diseases that are diagnosed using FISH include Prader-Willi syndrome, Angelman syndrome, chronic myelogenous leukemia, acute lymphoblastic leukemia, and Down syndrome. FISH can also be used to find sperm abnormalities and to evaluate remission of diseases.
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