title: New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.
abstract: Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; Electron Microscopy Core Facility, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schorb
authors.affiliation: Leica Microsystems (Schweiz) AG, Max Schmidheiny-Strasse 201, 9435 Heerbrugg, Switzerland.
authors.forename: Leander
authors.initials: L
authors.lastname: Gaechter
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Ori
authors.initials: O
authors.lastname: Avinoam
authors.affiliation: Leica Microsystems GmbH, Am Friedensplatz 3, 68165 Mannheim, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Sieckmann
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Mairi
authors.initials: M
authors.lastname: Clarke
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bebeacua
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Yury S
authors.initials: YS
authors.lastname: Bykov
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; Molecular Medicine Partnership Unit, EMBL/Universitätsklinikum Heidelberg, Heidelberg, Germany.
authors.forename: Andreas F-P
authors.initials: AF
authors.lastname: Sonnen
authors.affiliation: Leica Mikrosysteme GmbH, Hernalser Hauptstraße 219, 1170 Vienna, Austria.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lihl
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany; Molecular Medicine Partnership Unit, EMBL/Universitätsklinikum Heidelberg, Heidelberg, Germany. Electronic address: john.briggs@embl.de.
authors.forename: John A G
authors.initials: JAG
authors.lastname: Briggs
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D055032:Electron Microscope Tomography / Q000295:instrumentation* / Q000379:methods*; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D012984:Software
keywords: Correlative light and electron microscopy; Cryo-electron microscopy; Cryo-electron tomography; Cryo-fluorescence microscopy