title: Spiropyrans as molecular optical switches.
abstract: Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.
authors.affiliation: Applied Laser Physics and Laser Spectroscopy and Bielefeld Institute for Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Seefeldt
authors.forename: Robert
authors.initials: R
authors.lastname: Kasper
authors.forename: Mirco
authors.initials: M
authors.lastname: Beining
authors.forename: Jochen
authors.initials: J
authors.lastname: Mattay
authors.forename: Jutta
authors.initials: J
authors.lastname: Arden-Jacob
authors.forename: Norbert
authors.initials: N
authors.lastname: Kemnitzer
authors.forename: Karl Heinz
authors.initials: KH
authors.lastname: Drexhage
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Markus
authors.initials: M
authors.lastname: Sauer
mesh_terms: D001578:Benzopyrans / Q000737:chemistry* / Q000528:radiation effects; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry* / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D009574:Nitro Compounds / Q000737:chemistry* / Q000528:radiation effects; D038761:Photobleaching / Q000528:radiation effects; D011108:Polymers / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D001578:Benzopyrans; D005456:Fluorescent Dyes; D007211:Indoles; D009574:Nitro Compounds; D011108:Polymers; C088184:spiropyran