Biophysical Journal 72: 567-578 (1997)
© 1997 the Biophysical Society

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Time-Resolved Fluorescence Spectroscopy and Imaging of DNA Labeled with DAPI and Hoechst 33342 Using Three-Photon Excitation

Joseph R. Lakowicz ^*, Ignacy Gryczynski ^*, Henryk Malak ^*, Martin Schrader ^#, Peter Engelhardt , Hiroski Kano ^¶ and Stefan W. Hell ^¶

Center for Fluorescence Spectroscopy and Medical Biotechnology Center, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201 USA
Department of Medical Physics and Chemistry, University of Turku, FIN-20521 Turku, Finland
Department of Virology, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland
Centre for Biotechnology, University of Turku, FIN-20521 Turku, Finland

ABSTRACT

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-piperazinyl)) with two-photon (2h) and three-photon (3h) 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 and 3h 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 at 885 nm than for 2h 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.