title: Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging.
abstract: The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Yamanaka
authors.affiliation: Osaka University, The Institute of Scientific and Industrial Research, 8-1 Mihogaoka, Ibaraki, Osaka 565-0871, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Saito
authors.affiliation: Osaka University, Immunology Frontier Research Center, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Smith
authors.affiliation: Osaka University, The Institute of Scientific and Industrial Research, 8-1 Mihogaoka, Ibaraki, Osaka 565-0871, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Arai
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Uegaki
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yonemaru
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Mochizuki
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kawata
authors.affiliation: Osaka University, The Institute of Scientific and Industrial Research, 8-1 Mihogaoka, Ibaraki, Osaka 565-0871, Japan.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
authors.affiliation: Osaka University, Department of Applied Physics, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Fujita
mesh_terms: D004867:Equipment Design; D019544:Equipment Failure Analysis; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000295:instrumentation*; D008164:Luminescent Proteins / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation*; D057054:Molecular Imaging / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D008164:Luminescent Proteins