title: Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins.
abstract: The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon Unviersity, Pittsburgh PA 15213.|Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yan
authors.affiliation: Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Schwartz
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon Unviersity, Pittsburgh PA 15213.|Lane Center for Computational Biology, Carnegie Mellon University, Pittsburgh, PA 15213.
authors.forename: Suvrajit
authors.initials: S
authors.lastname: Maji
authors.affiliation: Department of Physics, University of New Mexico, Albuquerque, NM 87131.
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon Unviersity, Pittsburgh PA 15213.
authors.forename: Chris
authors.initials: C
authors.lastname: Szent-Gyorgyi
authors.affiliation: Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131.
authors.forename: Diane S
authors.initials: DS
authors.lastname: Lidke
authors.affiliation: Department of Physics, University of New Mexico, Albuquerque, NM 87131.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Lidke
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon Unviersity, Pittsburgh PA 15213.|Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213.|Lane Center for Computational Biology, Carnegie Mellon University, Pittsburgh, PA 15213.|Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213.
authors.forename: Marcel P
authors.initials: MP
authors.lastname: Bruchez
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012394:Rosaniline Dyes / Q000737:chemistry*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D012394:Rosaniline Dyes; C005095:malachite green
keywords: fluorescence; imaging agents; localization microscopy; proteins; super resolution