Multi-photon excitation microscopy involves simultaneous multi-photon absorption by a luminescent probe. Because the absorption rate depends on the square or cube of the intensity of the excitation source, a tightly focused laser beam enables three-dimensional spatial selectivity for excitation. For this reason, multi-photon excitation microscopy is a powerful tool for the three-dimensional imaging of cells, tissues and organs. In order to realize effective multi-photon excitation processes, a pulsed laser with a large peak intensity is required. On the one hand, the averaged power of excitation laser should be minimized to avoid phototoxicity. Therefore, a femtosecond (fs) pulsed Ti:sapphire laser has conventionally been employed as the excitation light source of multi-photon excitation microscopy.
Recently, several fs Yb-doped fiber lasers operating at 1030–1070 nm have been commercialized. The fs Yb-doped fiber laser is an attractive excitation light source for multi-photon excitation imaging. This is because the fiber laser oscillator is much smaller than that of a Ti:sapphire laser. Furthermore, the fs fiber laser is stable over wide temperature and humidity ranges, and requires less maintenance than a Ti:sapphire laser. However, there have been only a few reports on in vivo probes excitable by using a fs fiber lasers.
Our group has developed a salient two-photon absorption probe suitable for excitation using a fs fiber lasers , PY [(4,4’-((1E,1’E)-(3,8-dibutylpyrene-1,6-diyl)bis(ethene-2,1-diyl))bis(1-methylpyridin-1-ium) iodide)]. Maximum wavelength of fluorescence of PY in DMSO was 650 nm and the quantum yield was 0.8. Two-photon absorption maximum of PY was 950 nm with the cross-section of 1100 GM (1 GM = 10-50 cm4 per photon per molecule). Even at 1050 nm, the two-photon absorption cross-section was larger than 200 GM. PY exhibited sufficient water solubility (higher than 10-6) for staining a living cell and localized at mitochondria. Owing to the large fluorescent quantum yield and two-photon absorption cross-section of PY, HEK293 cell stained with PY showed a bright two-photon excitation microscope image even when a few mW of laser beam from a fs fiber laser was employed for excitation.
We have synthesized several fs fiber laser excitable two-photon absorption probes other than PY. In addition, three-photon absorption probes suitable to fs fiber laser excitation have been developed. The details of such probes will be discussed.
 Y. Niko, H. Moritomo, H. Sugihara, Y. Suzuki, J. Kawamata, and G. Konishi, J. Mater. Chem. B 3, 184 (2015).