Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser

We demonstrate that three-photon excitation images of both fixed and living biological specimens can be readily obtained using an all-solid-state Nd:YLF laser excitation source. Optically sectioned images of fixed Caenorhabditis elegans embryos stained with DAPI and embryos triple-labeled with DAPI, fluorescein and Texas Red are presented. Time series images of a living LLC-PK cell stained with Hoechst 33342 during the progression from metaphase to telophase are also presented. The mode of excitation was inferred from the power-law of anthracene and Hoechst 33342 fluorescence versus incident laser power and an axial resolution comparison of anthracene fluorescence with two-photon excited Calcium Crimson fluorescence. Multiphoton excitation imaging is an attractive method for optically sectioning live specimens because of the lower levels of phototoxicity produced compared to other optical sectioning techniques. The combination of two- and three-photon excitation extends the capabilities of a multiple- photon imaging system since a single wavelength can provide localized excitation of a wide variety of fluorophores whose collective emission spectra can span the entire visible spectrum.