title: A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.
abstract: Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction. Optimization of the intramolecular nucleophile and rhodamine-based fluorophore (electrophile) provide a suitable lifetime for the fluorescent open form, and equilibrium between the open form and the non-fluorescent closed form. We show that this spontaneously blinking fluorophore is suitable for single-molecule localization microscopy imaging deep inside cells and for tracking the motion of structures in living cells. We further demonstrate the advantages of this fluorophore over existing methodologies by applying it to nuclear pore structures located far above the coverslip with a spinning-disk confocal microscope and for repetitive time-lapse super-resolution imaging of microtubules in live cells for up to 1 h.
authors: Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Shin-Nosuke
authors: SN
authors: Uno
authors: Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Mako
authors: M
authors: Kamiya
authors: Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu 376-8515, Japan.
authors: Toshitada
authors: T
authors: Yoshihara
authors: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Ko
authors: K
authors: Sugawara
authors: 1] Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan [2] JST, PRESTO, Saitama 332-0012, Japan.
authors: Kohki
authors: K
authors: Okabe
authors: Center for International Research on Micronano Mechatronics, Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan.
authors: Mehmet C
authors: MC
authors: Tarhan
authors: Center for International Research on Micronano Mechatronics, Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan.
authors: Hiroyuki
authors: H
authors: Fujita
authors: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan.
authors: Takashi
authors: T
authors: Funatsu
authors: Laboratory for Cell Polarity Regulation, Quantitative Biology Center, RIKEN, Suita, 565-0874, Japan.
authors: Yasushi
authors: Y
authors: Okada
authors: Division of Molecular Science, Faculty of Science and Technology, Gunma University, Kiryu 376-8515, Japan.
authors: Seiji
authors: S
authors: Tobita
authors: 1] Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan [2] Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan [3] Basic Research Program, Japan Science and Technology Agency, Tokyo 102-0075, Japan.
authors: Yasuteru
authors: Y
authors: Urano
mesh_terms: D003500:Cyclization; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D010782:Photolysis; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013141:Spiro Compounds / Q000737:chemistry*; D059008:Time-Lapse Imaging
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D013141:Spiro Compounds