Multiphoton excitation of the DNA stains DAPI and Hoechst

,6-diamidino-2-phenylindole hydrochloride (DAPI) andHoechst 33342 were found to display two- or three-photon excitation from 810 to 910nm. We examined the effect of excitation wavelength on the mode of excitation forDAPI and Hoechst 33342 in the solvent isobutanol and when bound to double helicalDNA. For DAPI and Hoechst 33342 in isobutanol the mode of excitation changed fromtwo- to three-photon excitation over this wavelength range, with apparent transitionwavelengths of 855 and 880 nm, respectively. However, when bound to DNA, thetransition wavelength from two- to three-photon excitation increased for both probes.In the case of DAPI–DNA, the apparent transition wavelength increased to 868 nm,and three-photon excitation occurred above 900 nm. For Hoechst 33342–DNA themode of excitation was a mixture of two- and three-photon excitation to the longestexcitation wavelength of 910 nm, and we were unable to observe pure three-photonexcitation for Hoechst 33342–DNA. In the transition region the anisotropy of DAPIwas dependent on laser power, illustrating that the mode of excitation and transitionwavelengths will depend on the precise experimental conditions. Higher spatialresolution was observed for three-photon excitation of DAPI than for two-photonexcitation of Hoechst 33342. These results suggest that these probes can be used foreither two- or three-photon imaging of DNA or chromosomes.Keywords: fluorescence, spectroscopy, imaging, time-resolved fluorescence,two-photon excitation, three-photon excitation, DAPI, Hoechst 33342, two-photonmicroscopy