title: Axial superresolution via multiangle TIRF microscopy with sequential imaging and photobleaching.
abstract: We report superresolution optical sectioning using a multiangle total internal reflection fluorescence (TIRF) microscope. TIRF images were constructed from several layers within a normal TIRF excitation zone by sequentially imaging and photobleaching the fluorescent molecules. The depth of the evanescent wave at different layers was altered by tuning the excitation light incident angle. The angle was tuned from the highest (the smallest TIRF depth) toward the critical angle (the largest TIRF depth) to preferentially photobleach fluorescence from the lower layers and allow straightforward observation of deeper structures without masking by the brighter signals closer to the coverglass. Reconstruction of the TIRF images enabled 3D imaging of biological samples with 20-nm axial resolution. Two-color imaging of epidermal growth factor (EGF) ligand and clathrin revealed the dynamics of EGF-activated clathrin-mediated endocytosis during internalization. Furthermore, Bayesian analysis of images collected during the photobleaching step of each plane enabled lateral superresolution (<100 nm) within each of the sections.
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Yan
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Section on High-Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Peter W
authors.initials: PW
authors.lastname: Winter
authors.affiliation: Section on Biological Chemistry, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892;
authors.forename: Raul
authors.initials: R
authors.lastname: Rojas
authors.affiliation: Biomedical Imaging Research Services Section, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Victor
authors.initials: V
authors.lastname: Wang
authors.affiliation: Biomedical Imaging Research Services Section, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Matthew
authors.initials: M
authors.lastname: McAuliffe
authors.affiliation: Section on Biophotonics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892; pattersg@mail.nih.gov.
authors.forename: George H
authors.initials: GH
authors.lastname: Patterson
mesh_terms: D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D021621:Imaging, Three-Dimensional; D008856:Microscopy, Fluorescence / Q000379:methods; D038761:Photobleaching
chemical_list: D002966:Clathrin; D005456:Fluorescent Dyes; D004815:Epidermal Growth Factor
keywords: axial superresolution; photobleaching; total internal reflection fluorescence