title: In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate.
abstract: We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C(s) corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.
authors.affiliation: Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Barton
authors.forename: D
authors.initials: D
authors.lastname: Rhinow
authors.forename: A
authors.initials: A
authors.lastname: Walter
authors.forename: R
authors.initials: R
authors.lastname: Schröder
authors.forename: G
authors.initials: G
authors.lastname: Benner
authors.forename: E
authors.initials: E
authors.lastname: Majorovits
authors.forename: M
authors.initials: M
authors.lastname: Matijevic
authors.forename: H
authors.initials: H
authors.lastname: Niebel
authors.forename: H
authors.initials: H
authors.lastname: Müller
authors.forename: M
authors.initials: M
authors.lastname: Haider
authors.forename: M
authors.initials: M
authors.lastname: Lacher
authors.forename: S
authors.initials: S
authors.lastname: Schmitz
authors.forename: P
authors.initials: P
authors.lastname: Holik
authors.forename: W
authors.initials: W
authors.lastname: Kühlbrandt
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation* / Q000379:methods*; D016616:Cryoultramicrotomy; D005615:Freezing; D019617:Halobacterium salinarum / Q000648:ultrastructure; D046911:Macromolecular Substances / Q000032:analysis*; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods; D018717:Purple Membrane / Q000648:ultrastructure*; D055672:Static Electricity; D014027:Tobacco Mosaic Virus / Q000648:ultrastructure*
chemical_list: D046911:Macromolecular Substances