title: Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions.
abstract: We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics and ‡Department of Biochemistry, University of Oxford , Oxford, United Kingdom.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kaufmann
authors.forename: Pascale
authors.initials: P
authors.lastname: Schellenberger
authors.forename: Elena
authors.initials: E
authors.lastname: Seiradake
authors.forename: Ian M
authors.initials: IM
authors.lastname: Dobbie
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: Ilan
authors.initials: I
authors.lastname: Davis
authors.forename: Christoph
authors.initials: C
authors.lastname: Hagen
authors.forename: Kay
authors.initials: K
authors.lastname: Grünewald
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003080:Cold Temperature; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000032:analysis*; D005615:Freezing; D008164:Luminescent Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D058989:Vitrification
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins