Two-color two-photon excitation of fluorescence

Joseph R. Lakowicz, Ignacy Gryczynski, Henryk M. Malak, Zygmunt Gryczynski

Research output: Contribution to journalConference articlepeer-review

2 Scopus citations


We observed fluorescence emission from p-terphenyl, 2,5- diphenyl-1,3,4-oxadiazole (PPD) and indole resulting from two-photon excitation with two different wavelengths near 380 and 760 nm. For two-color two-photon (2C2P) excitation the emission spectra and intensity decays were the same as observed with single photon excitation with an equivalent energy near 250 nm. The two-color two-photon induced emission was observed when the samples were illuminated with both wavelengths, but only when the ps laser pulses were spatially and temporally overlapped. The signals were typically 50-fold to 1000-fold less for illumination at 380 or 760 nm alone. When illuminated with both wavelengths, and when both beams were simultaneously attenuated to the same extent, the emission intensity depended quadratically on the total illumination power, indicating two-photon excitation. When the illumination intensity at one wavelength was attenuated, the signal depended linearly on the power at each wavelength, indicating the participation of one-photon at each wavelength to the excitation process. For 2C2P excitation with both beams vertically polarized the time- zero anisotropies were larger than possible for single photon excitation. For PPD and p-terphenyl with intensity depended on the polarization of each beam in a manner consistent with co-linear transitions, but more complex behavior was found for indole. These results demonstrate that two-color two-photon excitation can be readily observed with modern ps laser sources. This phenomenon can have numerous applications in the chemical and biomedical sciences, as a method for spatial localization of the measured volume.

Original languageEnglish
Pages (from-to)368-380
Number of pages13
JournalProceedings of SPIE - The International Society for Optical Engineering
StatePublished - 1997
EventAdvances in Fluorescence Sensing Technology III - San Jose, CA, United States
Duration: 9 Feb 19979 Feb 1997


  • Anisotropy
  • Confocal microscopy
  • Fluorescence
  • Multi-photon excitation
  • Time-resolved fluorescence
  • Two-photon excitation


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