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.
Lakowicz
J R
JR
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA. jf@cfs.umbi.umd.edu
Gryczynski
I
I
Malak
H
H
Schrader
M
M
Engelhardt
P
P
Kano
H
H
Hell
S W
SW
D001562:Benzimidazoles; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002875:Chromosomes; D004247:DNA / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007211:Indoles; D007700:Kinetics; D007834:Lasers; D008027:Light; D008677:Metaphase; D008856:Microscopy, Fluorescence; D017785:Photons; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA; C017807:bisbenzimide ethoxide trihydrochloride