DAPI was first synthesised in 1971 in the laboratory of Otto Dann as part of a search for drugs to treat trypanosomiasis.

[3][4] DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample.

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.

This quantum-mechanical modeling has rationalized the absorption and fluorescence behavior given by minor groove binding and intercalation in the DNA pocket, in term of a reduced structural flexibility and polarization.