title: High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.
abstract: We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.
authors.affiliation: Department of Bioengineering, University of California, Berkeley, California, United States of America.
authors.forename: Liangxing
authors.initials: L
authors.lastname: Wu
authors.forename: Yingrui
authors.initials: Y
authors.lastname: Dai
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Jiang
authors.forename: Chutima
authors.initials: C
authors.lastname: Petchprayoon
authors.forename: Jessie E
authors.initials: JE
authors.lastname: Lee
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.forename: Yuling
authors.initials: Y
authors.lastname: Yan
authors.forename: Gerard
authors.initials: G
authors.lastname: Marriott
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D000873:Anthracenes / Q000737:chemistry; D001578:Benzopyrans / Q000737:chemistry*; D045744:Cell Line, Tumor; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D051381:Rats; D059010:Single-Cell Analysis; D013141:Spiro Compounds / Q000737:chemistry*; D016707:Tissue Fixation; D014230:Triazoles / Q000737:chemistry
chemical_list: D000873:Anthracenes; D001578:Benzopyrans; D005456:Fluorescent Dyes; D007211:Indoles; D013141:Spiro Compounds; D014230:Triazoles; C548873:merocyanine; C516131:spiro-merocyanine