title: Multimodal two-photon imaging using a second harmonic generation-specific dye.
abstract: Second harmonic generation (SHG) imaging can be used to visualize unique biological phenomena, but currently available dyes limit its application owing to the strong fluorescent signals that they generate together with SHG. Here we report the first non-fluorescent and membrane potential-sensitive SHG-active organic dye Ap3. Ap3 is photostable and generates SH signals at the plasma membrane with virtually no fluorescent signals, in sharp contrast to the previously used fluorescent dye FM4-64. When tested in neurons, Ap3-SHG shows linear membrane potential sensitivity and fast responses to action potentials, and also shows significantly reduced photodamage compared with FM4-64. The SHG-specific nature of Ap3 allows simultaneous and completely independent imaging of SHG signals and fluorescent signals from various reporter molecules, including markers of cellular organelles and intracellular calcium. Therefore, this SHG-specific dye enables true multimodal two-photon imaging in biological samples.
authors.affiliation: Department of Pharmacology School of Medicine, Keio University, 35 Shinanomachi, Tokyo 160-8582, Japan.|Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan.
authors.forename: Mutsuo
authors.initials: M
authors.lastname: Nuriya
authors.affiliation: Graduate School of Pure and Applied Sciences and Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Tsukuba 305-8577, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Fukushima
authors.affiliation: Graduate School of Pure and Applied Sciences and Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Tsukuba 305-8577, Japan.
authors.forename: Atsuya
authors.initials: A
authors.lastname: Momotake
authors.affiliation: Department of Pharmacology School of Medicine, Keio University, 35 Shinanomachi, Tokyo 160-8582, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Shinotsuka
authors.affiliation: Department of Pharmacology School of Medicine, Keio University, 35 Shinanomachi, Tokyo 160-8582, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Yasui
authors.affiliation: Graduate School of Pure and Applied Sciences and Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Tsukuba 305-8577, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Arai
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000648:ultrastructure*; D004396:Coloring Agents; D003412:Cricetulus; D008564:Membrane Potentials; D064847:Multimodal Imaging; D009474:Neurons / Q000648:ultrastructure*; D017785:Photons; D066244:Proton Magnetic Resonance Spectroscopy; D000073798:Second Harmonic Generation Microscopy / Q000379:methods*; D013050:Spectrometry, Fluorescence
chemical_list: D004396:Coloring Agents