Biophysical Journal, Vol 72, 567-578, Copyright © 1997 by       

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

       JR Lakowicz, I Gryczynski, H Malak, M Schrader, P Engelhardt, H Kano and SW Hell

       Department of Biochemistry and Molecular Biology, University of Maryland School of
       Medicine, Baltimore 21201, USA.
       jf@cfs.umbi.umd.edu


       We e amined 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 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-ph ton 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 excitat on of DAPI-stained chromosomes at 970 nm was 
       demonstrated by the power dependence in the fluorescence microscope.