DAPI (pronounced "DAPPY"), or 4 ', 6-diamidino-2-phenylindole, is a fluorescent dye that binds strongly to adenine-thymine-rich regions in DNA. It is widely used in fluorescence microscopy. Because DAPI can penetrate intact cell membranes, it can be used to stain living and fixed cells, although DAPI is less efficient at crossing membranes in living cells and thus provides a marker of membrane viability.
Figure 1. Chemical structure of DAPI.
DAPI was first synthesized in Otto Dann's laboratory in 1971 as part of a search for drugs to treat trypanosomiasis. Although unsuccessful as a drug, further research has shown that it binds strongly to DNA and fluoresces when bound. This led to its use in ultracentrifugation in 1975 to identify mitochondrial DNA, the first recorded use of DAPI as a fluorescent DNA stain. The strong fluorescence when combined with DNA caused DAPI to be quickly used for fluorescence staining of DNA by fluorescence microscopy. It was proved to be useful for detecting DNA in plants, metazoans and bacterial cells, as well as virus particles in the late 1970s, and its quantitative staining of intracellular DNA in 1977. At the same time, the use of DAPI as a DNA stain for flow cytometry has also been demonstrated.
When bound to double-stranded DNA, DAPI has maximum absorption at a wavelength of 358 nm (ultraviolet) and its maximum emission at 461 nm (blue). Therefore, for fluorescence microscopy, DAPI is excited by ultraviolet light and detected by a blue/cyan filter. The emission peak is quite broad. DAPI can also bind to RNA, although it is not very fluorescent. When combined with RNA, its emission moves to around 500 nm. DAPI's blue emission is perfect for microscopyists who want to use multiple fluorescent dyes in a single sample. There is some fluorescence overlap between DAPI and green fluorescent molecules such as fluorescein and green fluorescent protein (GFP), but this effect is small. If very accurate image analysis is required, then using spectral decomposition can solve this problem. In addition to analytical fluorescence microscopy, DAPI is also commonly used to label cell cultures to detect DNA from contaminating mycoplasma or viruses. Once stained with DAPI, the labeled mycoplasma or virus particles in the growth medium fluoresce, making them easy to detect.
Figure 2. DAPI (magenta) bound to the minor groove of DNA (green and blue).
DAPI can be used for fixed cell staining. The concentration of DAPI required for live cell staining is usually high. It is rarely used in living cells. It is labeled non-toxic in its MSDS, and although it has not been shown to be mutagenic to E. coli, it is labeled as a known mutagen in the manufacturer's information. Because it is a DNA-binding compound, it may have low levels of mutagenic properties, and care should be taken during handling and disposal.
Figure 3. Endothelial cells stained with DAPI (blue), phalloidin (red) and through immunofluorescence via an antibody bound to fluorescein isothiocyanate (FITC) (green).