title: Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies.
abstract: A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand.
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Cazaux
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Anaïs
authors.initials: A
authors.lastname: Sadoun
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Biarnes-Pelicot
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Manuel
authors.initials: M
authors.lastname: Martinez
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Sameh
authors.initials: S
authors.lastname: Obeid
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France; APHM, Hôpital de la Conception, Laboratoire d'Immunologie, Marseille F-13385, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bongrand
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Limozin
authors.affiliation: Aix Marseille Université, LAI UM 61, Marseille F-13288, France; Inserm, UMR_S 1067, Marseille F-13288, France; CNRS, UMR 7333, Marseille F-13288, France. Electronic address: pierre-henri.puech@inserm.fr.
authors.forename: Pierre-Henri
authors.initials: PH
authors.lastname: Puech
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D020013:Calcium Signaling / Q000276:immunology; D002460:Cell Line; D018914:Cells, Immobilized / Q000276:immunology; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000276:immunology*; D051379:Mice; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D059010:Single-Cell Analysis; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014966:Xanthenes; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D005456:Fluorescent Dyes; D014966:Xanthenes; D020744:rac GTP-Binding Proteins
keywords: Activation; Atomic force microscopy; Cell mechanics; Fluorescence microscopy; Immune cells; Signalling