title: Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins.
abstract: We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: Errin
authors.initials: E
authors.lastname: Johnson
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: Elena
authors.initials: E
authors.lastname: Seiradake
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Ilan
authors.initials: I
authors.lastname: Davis
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: Kay
authors.initials: K
authors.lastname: Grünewald
authors.affiliation: 1] Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK [2] Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kaufmann
mesh_terms: D002460:Cell Line; D006657:Histones; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D008854:Microscopy, Electron / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D006657:Histones; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins
references.citation: Nat Methods. 2011 Dec;8(12):1047-9
references.pmid: 21983925
references.citation: Nat Methods. 2014 Jul;11(7):737-9
references.pmid: 24813625
references.citation: J Cell Biol. 2011 Jan 10;192(1):111-9
references.pmid: 21200030
references.citation: Nat Methods. 2009 Jan;6(1):24-32
references.pmid: 19116611
references.citation: Science. 2008 Apr 11;320(5873):246-9
references.pmid: 18292304
references.citation: Nat Methods. 2008 Nov;5(11):943-5
references.pmid: 18794861
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):6136-41
references.pmid: 22474357
references.citation: J Cell Biol. 1976 Sep;70(3):608-21
references.pmid: 783172
references.citation: Nat Methods. 2012 Oct;9(10):1005-12
references.pmid: 22961245
references.citation: J Microsc. 2003 Oct;212(Pt 1):53-61
references.pmid: 14516362
references.citation: Nat Methods. 2011 Jun;8(6):499-508
references.pmid: 21552254
references.citation: Nat Commun. 2014;5:3992
references.pmid: 24886825
references.citation: Traffic. 2009 Feb;10(2):131-6
references.pmid: 19054388
references.citation: Microsc Res Tech. 2001 May 15;53(4):313-21
references.pmid: 11340677
references.citation: Histochem J. 1989 May;21(5):249-58
references.pmid: 2476415
references.citation: Histochem J. 1980 Jul;12(4):381-403
references.pmid: 7440248
references.citation: Nat Protoc. 2014 Oct;9(10):2382-94
references.pmid: 25211515
references.citation: Nat Methods. 2011 Jan;8(1):80-4
references.pmid: 21102453
references.citation: J Microsc. 2011 Apr;242(1):46-54
references.pmid: 21118230
references.citation: Microsc Microanal. 2010 Feb;16(1):64-72
references.pmid: 20082730
references.citation: J Histochem Cytochem. 2009 Dec;57(12):1103-12
references.pmid: 19654103
references.citation: Nat Struct Mol Biol. 2010 Apr;17(4):398-402
references.pmid: 20228801
references.citation: J Cell Sci. 2014 Oct 15;127(Pt 20):4351-5
references.pmid: 25146397
references.citation: Nat Methods. 2012 Feb;9(2):152-8
references.pmid: 22290187
references.citation: J Microsc. 2010 Jan;237(1):23-38
references.pmid: 20055916
references.citation: Genes Dev. 2010 Jul 1;24(13):1351-63
references.pmid: 20551166
references.citation: Biophys J. 2006 Dec 1;91(11):4258-72
references.pmid: 16980368
references.citation: Nano Lett. 2014 Jul 9;14(7):4171-5
references.pmid: 24884378
references.citation: J Cell Biol. 1963 Apr;17:208-12
references.pmid: 13986422
references.citation: PLoS One. 2013;8(10):e77209
references.pmid: 24204771
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20308-13
references.pmid: 18077327
references.citation: Methods Cell Biol. 2012;111:157-73
references.pmid: 22857928
references.citation: PLoS One. 2012;7(1):e30826
references.pmid: 22292051
references.citation: PLoS One. 2013;8(4):e60877
references.pmid: 23577174
references.citation: Curr Opin Chem Biol. 2014 Jun;20:22-8
references.pmid: 24793374
references.citation: Ultramicroscopy. 2014 Aug;143:3-14
references.pmid: 24637200
references.citation: Curr Opin Chem Biol. 2014 Jun;20:86-91
references.pmid: 24951858
references.citation: Methods Cell Biol. 2012;111:325-56
references.pmid: 22857936
references.citation: Opt Lett. 1994 Jun 1;19(11):780-2
references.pmid: 19844443
references.citation: Eur J Neurosci. 2008 Dec;28(12):2365-70
references.pmid: 19087167
references.citation: Nat Methods. 2006 Oct;3(10):793-5
references.pmid: 16896339
references.citation: Histochem Cell Biol. 2014 Jun;141(6):629-38
references.pmid: 24522395
references.citation: Nat Commun. 2013;4:2244
references.pmid: 23921440
references.citation: Nat Methods. 2008 Nov;5(11):947-9
references.pmid: 18794860
references.citation: Curr Opin Cell Biol. 2007 Oct;19(5):543-50
references.pmid: 17935964
references.citation: PLoS One. 2012;7(2):e31128
references.pmid: 22319608
references.citation: J Microsc. 2000 May;198(Pt 2):82-7
references.pmid: 10810003
references.citation: Methods Mol Biol. 2014;1117:559-92
references.pmid: 24357380
references.citation: Nat Cell Biol. 2012 Dec;14(12):1305-13
references.pmid: 23178881
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14271-6
references.pmid: 18796604
references.citation: Science. 2006 Sep 15;313(5793):1642-5
references.pmid: 16902090
references.citation: J Microsc. 2007 May;226(Pt 2):182-9
references.pmid: 17444947
references.citation: J Cell Biol. 2014 Feb 3;204(3):409-22
references.pmid: 24469634
references.citation: J Struct Biol. 2014 May;186(2):205-13
references.pmid: 24698954