Three-photon excitation in fluorescence microscopy

Stefan W. Hell, Karsten Bahlmann, Martin Schrader, Aleksi Soini, Henryk Malak, Ignacy Gryczynski, Joseph R. Lakowicz

Research output: Contribution to journalArticlepeer-review

132 Scopus citations


We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium-sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

Original languageEnglish
Pages (from-to)71-74
Number of pages4
JournalJournal of Biomedical Optics
Issue number1
StatePublished - Jan 1996


  • Fluorescence microscopy
  • Resolution
  • Three-photon excitation


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