Multiphoton excitation of the DNA stains DAPI and Hoechst

The DNA stains 40′ ,6-diamidino-2-phenylindole hydrochloride (DAPI) and Hoechst 33342 were found to display two- or three-photon excitation from 810 to 910 nm. We examined the effect of excitation wavelength on the mode of excitation for DAPI and Hoechst 33342 in the solvent isobutanol and when bound to double helical DNA. For DAPI and Hoechst 33342 in isobutanol the mode of excitation changed from two- to three-photon excitation over this wavelength range, with apparent transition wavelengths of 855 and 880 nm, respectively. However, when bound to DNA, the transition 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 the mode of excitation was a mixture of two- and three-photon excitation to the longest excitation wavelength of 910 nm, and we were unable to observe pure three-photon excitation for Hoechst 33342-DNA. In the transition region the anisotropy of DAPI was dependent on laser power, illustrating that the mode of excitation and transition wavelengths will depend on the precise experimental conditions. Higher spatial resolution was observed for three-photon excitation of DAPI than for two-photon excitation of Hoechst 33342. These results suggest that these probes can be used for either two- or three-photon imaging of DNA or chromosomes.