Jun Kawamata1 Yasutaka Suzuki1

1, Yamaguchi University, Yamaguchi, , Japan

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 [1], 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.

[1] Y. Niko, H. Moritomo, H. Sugihara, Y. Suzuki, J. Kawamata, and G. Konishi, J. Mater. Chem. B 3, 184 (2015).