Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation.

We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5'-bi'1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi nyl)) with two-photon (2h nu) and three-photon (3h nu) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three-photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2h nu and 3h nu excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3h nu at 885 nm than for 2h nu at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.