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In-Focus Electrostatic Zach Phase Plate Imaging for Transmission Electron Microscopy with Tunable Phase Contrast of Frozen Hydrated Biological Samples

Published online by Cambridge University Press:  02 January 2014

Nicole Frindt ,
Marco Oster ,
Simon Hettler ,
Björn Gamm ,
Levin Dieterle ,
Wolfgang Kowalsky ,
Dagmar Gerthsen  and
Rasmus R. Schröder
Nicole Frindt*
Affiliation:
CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
Marco Oster
Affiliation:
CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
Simon Hettler
Affiliation:
Laboratorium für Elektronenmikroskopie, Karlsruhe Institut für Technologie (KIT), Engesserstr 7, 76128 Karlsruhe, Germany
Björn Gamm
Affiliation:
Laboratorium für Elektronenmikroskopie, Karlsruhe Institut für Technologie (KIT), Engesserstr 7, 76128 Karlsruhe, Germany
Levin Dieterle
Affiliation:
Institut für Hochfrequenztechnik, Technische Universität Braunschweig, Schleinitzstr 22, 38106 Braunschweig, Germany
Wolfgang Kowalsky
Affiliation:
Institut für Hochfrequenztechnik, Technische Universität Braunschweig, Schleinitzstr 22, 38106 Braunschweig, Germany Innovation Lab GmbH, Heidelberg, Speyerer Str. 4, 69115 Heidelberg, Germany
Dagmar Gerthsen
Affiliation:
Laboratorium für Elektronenmikroskopie, Karlsruhe Institut für Technologie (KIT), Engesserstr 7, 76128 Karlsruhe, Germany
Rasmus R. Schröder*
Affiliation:
CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany Innovation Lab GmbH, Heidelberg, Speyerer Str. 4, 69115 Heidelberg, Germany CAM Centre of Advanced Materials, Universität Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
*
*Corresponding author. E-mail: [email protected]
**Corresponding author. E-mail: [email protected]
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Abstract

Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

Keywords

electrostatic phase platecryo transmission electron microscopyweak-phase objectphase contrastfrozen hydrated biological samplescryo samplessingle-sideband effectchargingcontamination
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 1 , February 2014 , pp. 175 - 183
Copyright
Copyright © Microscopy Society of America 2014 

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References

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. (2008). Molecular Biology of the Cell, 5th ed. New York: Garland Science Taylor & Francis Group.Google Scholar
Boersch, H. (1947). Über die Kontraste von Atomen im Elektronenmikroskop. Z Naturforschung 2a, 615–633.Google Scholar
Buijsse, B., Van Laarhoven, F.M.H.M., Schmid, A.K., Cambie, R., Cabrini, S., Jin, J. & Glaeser, R.M. (2011). Design of a hybrid double-sideband/single-sideband (schlieren) objective aperture suitable for electron microscopy. Ultramicroscopy 111, 1688–1695.Google Scholar
Cambie, R., Downing, K.H., Typke, D., Glaeser, R.M. & Jin, J. (2007). Design of a microfabricated, two-electrode phasecontrast element suitable for electron microscopy. Ultramicroscopy 107, 329–339.Google Scholar
Danev, R. & Nagayama, K. (2001). Transmission electron microscopy with Zernike phase plate. Ultramicroscopy 88, 243–252.CrossRefGoogle ScholarPubMed
Danev, R. & Nagayama, K. (2008). Single particle analysis based on Zernike phase contrast transmission electron microscopy. J Struct Biol 161, 211–218.Google Scholar
Danev, R. & Nagayama, K. (2011). Optimizing the phase shift and the cut-on periodicity of phase plates for TEM. Ultramicroscopy 111, 1305–1315.Google Scholar
Danev, R., Okawara, H., Usuda, N., Kametani, K. & Nagayama, K. (2002). A novel phase-contrast transmission electron microscopy producing high-contrast topographic images of weak objects. J Biol Phys 28, 627–635.Google Scholar
Dubochet, J., Lepault, J., Freeman, R., Berriman, J.A. & Homo, J.C. (1982). Electron microscopy of frozen water and aqueous solutions. J Microsc 128, 219–237.Google Scholar
Frindt, N. (2013). Development and implementation of electrostatic Zach phase plates for phase contrast transmission electron microscopy. PhD thesis. Universität Heidelberg, Heidelberg, Germany. Google Scholar
Gamm, B., Dries, M., Schultheiss, K., Blank, H., Rosenauer, A., Schröder, R.R. & Gerthsen, D. (2010). Object wave reconstruction by phase-plate transmission electron microscopy. Ultramicroscopy 110, 807–814.Google Scholar
Glaeser, R.M., Sassolini, S., Cambie, R., Jin, J., Cabrini, S., Schmid, A., Danev, R., Buijsse, B., Csencsits, R., Downing, K.H., Larson, D.M., Typke, D. & Han, B.G. (2013). Minimizing electrostatic charging of an aperture used to produce in-focus phase contrast in the TEM. Ultramicroscopy 135, 6–15.CrossRefGoogle ScholarPubMed
Hettler, S., Gamm, B., Dries, M., Frindt, N., Schröder, R.R. & Gerthsen, D. (2012). Improving fabrication and application of Zach phase plates for phase-contrast transmission electron microscopy. Microsc Microanal 18, 1010–1015.CrossRefGoogle ScholarPubMed
Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44–49.CrossRefGoogle ScholarPubMed
Majorovits, E., Barton, B., Schultheiss, K., Pérez-Willard, F., Gerthsen, D. & Schröder, R.R. (2007). Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate. Ultramicroscopy 107, 213–226.Google Scholar
Matsumoto, T. & Tonomura, A. (1996). The phase constancy of electron waves traveling through Boerschs electrostatic phase plate. Ultramicroscopy 63, 5–10.Google Scholar
Murata, K., Liu, X., Danev, R., Jakana, J., Schmid, M.F., King, J., Nagayama, K. & Chiu, W. (2010). Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and sub-nanometer resolutions. Structure 18, 903–912.CrossRefGoogle Scholar
Nagayama, K. (2008). Development of phase plates for electron microscopes and their biological application. Eur Biophys J 37, 345–358.CrossRefGoogle ScholarPubMed
Reimer, L. & Kohl, H. (2008). Transmission Electron Microscopy: Physics of Image Formation. New York: Springer Verlag.Google Scholar
Schultheiss, K., Pérez-Willard, F., Barton, B., Gerthsen, D. & Schröder, R.R. (2006). Fabrication of a Boersch phase plate for phase contrast imaging in a transmission electron microscope. Rev Sci Instrum 77, 033701. CrossRefGoogle Scholar
Schultheiss, K., Zach, J., Gamm, B., Dries, M., Frindt, N., Schröder, R.R. & Gerthsen, D. (2010). New electrostatic phase plate for transmission electron microscopy and its application for wave-function reconstruction. Microsc Microanal 16, 536–537.Google Scholar
Vitos, L., Ruban, A.V., Skriver, H.L. & Kollár, J. (1998). The surface energy of metals. Surf Sci 411, 186–202.CrossRefGoogle Scholar
Walker, M., Nordquist, C., Czaplewski, D., Patrizi, G., McGruer, N. & Krim, J. (2010). Impact of in situ oxygen plasma cleaning on the resistance of Ru and Au-Ru based rf microelectromechanical system contacts in vacuum. J Appl Phys 107, 084509. CrossRefGoogle Scholar
Weber, L., Lehr, M. & Gmelin, E. (1996). Investigation of the transport properties of gold point contacts. Physica B Condens Matter 217, 181–192.Google Scholar
Zach, J. (2008). Phase plate, image producing method and electron microscope. PCT Patent WO/2008/061603; EPO Patent 1989723-A2, European Patent Office, Munich. Google Scholar