title: Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells.
abstract: Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure.
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Peddie
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Ken
authors.initials: K
authors.lastname: Blight
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Wilson
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Melia
authors.affiliation: Department of Biology, The University of York, Heslington, York, UK.
authors.forename: Jo
authors.initials: J
authors.lastname: Marrison
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Carzaniga
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Marie-Charlotte
authors.initials: MC
authors.lastname: Domart
authors.affiliation: Department of Biology, The University of York, Heslington, York, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: O'Toole
authors.affiliation: Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK; Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
authors.forename: Banafshe
authors.initials: B
authors.lastname: Larijani
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Collinson
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000378:metabolism*; D005453:Fluorescence; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008322:Mammals / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009685:Nuclear Envelope / Q000378:metabolism; D056489:Nucleoplasmins / Q000378:metabolism
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D056489:Nucleoplasmins; D049452:Green Fluorescent Proteins
keywords: Correlative light and electron microscopy; Diacylglycerol; GFP; In-resin fluorescence; Integrated light and electron microscopy; Mammalian cells