Journal of Fluorescence

, Volume 4, Issue 4, pp 331–336 | Cite as

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

  • Ignacy Gryczynski
  • Joseph R. Lakowicz
Fluorescence Imaging and Microscopy


We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at −20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.

Key Words

Hoechst 33342 one-photon excitation two-photon excitation anisotropy decay time-resolved fluorescence frequency-domain DNA 

Abbreviations used




4′,6-diamidino-2-phenylindole, hydrochloride


Hoechst 33342, bis-benzimide, 2,5′-bi-1H-benzimidazole, 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)


one-photon excitation


two-photon excitation


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Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • Ignacy Gryczynski
    • 1
  • Joseph R. Lakowicz
    • 1
  1. 1.Center for Fluorescence Spectroscopy, Department of Biological ChemistryUniversity of Maryland School of MedicineBaltimore

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