title: Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.
abstract: Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.
authors.affiliation: UMR144 CNRS/Institut Curie, 75005 Paris, France; jerome.boulanger@curie.fr salamero@curie.fr.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Boulanger
authors.affiliation: Plateforme Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie Institut Curie, 75005 Paris, France; Roper Scientific SAS, 91017 Evry, France; and.
authors.forename: Charles
authors.initials: C
authors.lastname: Gueudry
authors.affiliation: Plateforme Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie Institut Curie, 75005 Paris, France; Roper Scientific SAS, 91017 Evry, France; and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Münch
authors.affiliation: UMR144 CNRS/Institut Curie, 75005 Paris, France;
authors.forename: Bertrand
authors.initials: B
authors.lastname: Cinquin
authors.affiliation: UMR144 CNRS/Institut Curie, 75005 Paris, France; Plateforme Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie Institut Curie, 75005 Paris, France;
authors.forename: Perrine
authors.initials: P
authors.lastname: Paul-Gilloteaux
authors.affiliation: UMR144 CNRS/Institut Curie, 75005 Paris, France;
authors.forename: Sabine
authors.initials: S
authors.lastname: Bardin
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CNRS/Commissariat à l'Energie Atomique/Institut National de la Recherche Agronomique/Université Joseph Fourier, Grenoble 38054, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Guérin
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CNRS/Commissariat à l'Energie Atomique/Institut National de la Recherche Agronomique/Université Joseph Fourier, Grenoble 38054, France.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Senger
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CNRS/Commissariat à l'Energie Atomique/Institut National de la Recherche Agronomique/Université Joseph Fourier, Grenoble 38054, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Blanchoin
authors.affiliation: UMR144 CNRS/Institut Curie, 75005 Paris, France; Plateforme Imagerie Cellulaire et Tissulaire-Infrastructure en Biologie Santé et Agronomie Institut Curie, 75005 Paris, France; jerome.boulanger@curie.fr salamero@curie.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Salamero
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D037241:Mannose-Binding Lectins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D058105:Polymerization; D015203:Reproducibility of Results; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein; D000099193:rab11 GTP-Binding Proteins
chemical_list: D000199:Actins; D015703:Antigens, CD; D049452:Green Fluorescent Proteins; D037181:Lectins, C-Type; D008164:Luminescent Proteins; D037241:Mannose-Binding Lectins; D020691:rab GTP-Binding Proteins; D000097573:Red Fluorescent Protein; D000099193:rab11 GTP-Binding Proteins; C121916:CD207 protein, human
keywords: 3D image reconstruction; TIRFM; high resolution; living cells; membrane recycling