title: Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.
abstract: Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.
authors.affiliation: Department of Bioengineering, School of Engineering, Santa Clara University, 500 EI Camino Real, Santa Clara, CA 95053, USA.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Yan
authors.forename: Chutima
authors.initials: C
authors.lastname: Petchprayoon
authors.forename: Shu
authors.initials: S
authors.lastname: Mao
authors.forename: Gerard
authors.initials: G
authors.lastname: Marriott
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001578:Benzopyrans / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D007211:Indoles / Q000737:chemistry*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D061848:Optical Imaging; D010078:Oxazines / Q000737:chemistry; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D013141:Spiro Compounds / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000199:Actins; D000906:Antibodies; D001578:Benzopyrans; D005456:Fluorescent Dyes; D007211:Indoles; D010078:Oxazines; D018343:Receptors, Adrenergic, beta-2; D013141:Spiro Compounds; C548873:merocyanine; C560447:spironaphthoxazine