DAPI
Encyclopedia
DAPI or 4',6-diamidino-2-phenylindole is a fluorescent
stain
that binds strongly to A-T rich regions in DNA
. It is used extensively in fluorescence microscopy. DAPI can pass through an intact cell membrane
therefore it can be used to stain both live and fixed
cells, though it passes through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower.
although it was unsuccessful as a drug. Further investigation indicated it bound strongly to DNA and became more fluorescent when it did so. This led to its use in identifying mitochondrial DNA in ultracentrifugation in 1975, the first recorded use of DAPI as a fluorescent DNA stain.
Strong fluorescence when bound to DNA led to the rapid adoption of DAPI for fluorescent staining of DNA for fluorescence microscopy. Its use for detecting DNA in plant
, metazoa and bacteria
cells and virus
particles was demonstrated in the late 1970s, and quantitative staining of DNA inside cells was demonstrated in 1977. Use of DAPI as a DNA stain for flow cytometry
was also demonstrated around this time.
DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet
) and its emission maximum is at 461 nm (blue
). Therefore for fluorescence microscopy DAPI is excited with ultraviolet
light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA
, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.
DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample. There is some fluorescence overlap between DAPI and green-fluorescent molecules like fluorescein
and green fluorescent protein
(GFP) but the effect of this is small. Use of spectral unmixing can account for this effect if extremely precise image analysis is required.
Outside of analytical fluorescence light microscopy DAPI is also popular for labeling of cell cultures to detect the DNA of contaminating mycoplasma
or virus
. The labelled mycoplasma or virus particles in the growth medium
fluoresce once stained by DAPI making them easy to detect.
The Hoechst stain
s are similar to DAPI in that they are also blue-fluorescent DNA stains which are compatible with both live- and fixed-cell applications.
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
stain
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...
that binds strongly to A-T rich regions in DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. It is used extensively in fluorescence microscopy. DAPI can pass through an intact cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
therefore it can be used to stain both live and fixed
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...
cells, though it passes through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower.
History
DAPI was first synthesised in 1971 in the laboratory of Otto Dann as part of a search for drugs to treat trypanosomiasisTrypanosomiasis
Trypanosomiasis or trypanosomosis is the name of several diseases in vertebrates caused by parasitic protozoan trypanosomes of the genus Trypanosoma. Approximately 500,000 men, women and children in 36 countries of sub-Saharan Africa suffer from human African trypanosomiasis which is caused by...
although it was unsuccessful as a drug. Further investigation indicated it bound strongly to DNA and became more fluorescent when it did so. This led to its use in identifying mitochondrial DNA in ultracentrifugation in 1975, the first recorded use of DAPI as a fluorescent DNA stain.
Strong fluorescence when bound to DNA led to the rapid adoption of DAPI for fluorescent staining of DNA for fluorescence microscopy. Its use for detecting DNA in plant
Plant
Plants are living organisms belonging to the kingdom Plantae. Precise definitions of the kingdom vary, but as the term is used here, plants include familiar organisms such as trees, flowers, herbs, bushes, grasses, vines, ferns, mosses, and green algae. The group is also called green plants or...
, metazoa and bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
cells and virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
particles was demonstrated in the late 1970s, and quantitative staining of DNA inside cells was demonstrated in 1977. Use of DAPI as a DNA stain for flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
was also demonstrated around this time.
Fluorescence properties
When bound to double-stranded DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
) and its emission maximum is at 461 nm (blue
Blue
Blue is a colour, the perception of which is evoked by light having a spectrum dominated by energy with a wavelength of roughly 440–490 nm. It is considered one of the additive primary colours. On the HSV Colour Wheel, the complement of blue is yellow; that is, a colour corresponding to an equal...
). Therefore for fluorescence microscopy DAPI is excited with ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...
light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
and green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP) but the effect of this is small. Use of spectral unmixing can account for this effect if extremely precise image analysis is required.
Outside of analytical fluorescence light microscopy DAPI is also popular for labeling of cell cultures to detect the DNA of contaminating mycoplasma
Mycoplasma
Mycoplasma refers to a genus of bacteria that lack a cell wall. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans,...
or virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
. The labelled mycoplasma or virus particles in the growth medium
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
fluoresce once stained by DAPI making them easy to detect.
Live cells and toxicity
DAPI can be used for both fixed and live cell staining, though the concentration of DAPI needed for live cell staining is generally much higher than for fixed cells. It is labeled non-toxic in its MSDS and though was not shown to have mutagenicity to E. coli, it is labelled as a known mutagen in manufacturer information. As it is a DNA binding compound it is likely to have some low level mutagenic properties and care should be taken in its handling and disposal.Alternatives
Hoechst stain
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains:...
s are similar to DAPI in that they are also blue-fluorescent DNA stains which are compatible with both live- and fixed-cell applications.