title: Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell.
abstract: We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed- and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.
authors.affiliation: Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, 37070 Göttingen, Germany.
authors.forename: Birka
authors.initials: B
authors.lastname: Hein
authors.forename: Katrin I
authors.initials: KI
authors.lastname: Willig
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival; D002477:Cells / Q000166:cytology*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000191:economics / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D048328:Potoroidae
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria
references.citation: Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8206-10
references.pmid: 10899992
references.citation: Opt Lett. 1994 Jun 1;19(11):780-2
references.pmid: 19844443
references.citation: Angew Chem Int Ed Engl. 2007;46(33):6266-70
references.pmid: 17640007
references.citation: Science. 2008 Feb 8;319(5864):810-3
references.pmid: 18174397
references.citation: Science. 1990 Apr 6;248(4951):73-6
references.pmid: 2321027
references.citation: Biophys J. 2001 Apr;80(4):2029-36
references.pmid: 11259316
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: Science. 2008 Apr 11;320(5873):246-9
references.pmid: 18292304
references.citation: Phys Rev Lett. 2005 Apr 15;94(14):143903
references.pmid: 15904066
references.citation: Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17565-9
references.pmid: 16314572
references.citation: Nat Methods. 2008 May;5(5):417-23
references.pmid: 18408726
references.citation: J Biol Chem. 2001 Aug 3;276(31):29188-94
references.pmid: 11387331
references.citation: Biophys J. 2006 Dec 1;91(11):4258-72
references.pmid: 16980368
references.citation: Science. 2007 May 25;316(5828):1153-8
references.pmid: 17525330
references.citation: Nat Methods. 2007 Nov;4(11):915-8
references.pmid: 17952088
references.citation: Science. 2006 Sep 15;313(5793):1642-5
references.pmid: 16902090
references.citation: Nat Methods. 2006 Oct;3(10):793-5
references.pmid: 16896339
references.citation: Nat Methods. 2006 Sep;3(9):721-3
references.pmid: 16896340