title: Integrated light and scanning electron microscopy of GFP-expressing cells.
abstract: Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes.
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Peddie
authors.affiliation: Department of Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
authors.forename: Nalan
authors.initials: N
authors.lastname: Liv
authors.affiliation: Department of Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands.
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Hoogenboom
authors.affiliation: Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, United Kingdom.
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Collinson
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D015925:Cryopreservation; D004075:Diglycerides / Q000378:metabolism; D004285:Dogs; D022001:Focal Adhesions / Q000648:ultrastructure*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008855:Microscopy, Electron, Scanning / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008867:Microtomy; D051419:Paxillin / Q000096:biosynthesis; D016613:Plastic Embedding; D011554:Pseudopodia / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D004075:Diglycerides; D051419:Paxillin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: CLEM; Correlative; Cryo-fluorescence; Green fluorescent protein; ILEM; ILSEM; In-resin fluorescence; Integrated