Phase Plate Electron Microscopy

Conference paper


An electron microscope enhanced with phase plates has practical advantages, particular for biological electron microscopy. It permits collecting high-contrast images of close-to-life biological structures with cryo-fixation and without harsh sample preparations including staining. Here we describe the state of the art of phase plate electron microscopy. Focuses are given to methodological aspects and biological applications with two dimensional and three-dimensional imaging.


Electron microscopy Tomography Phase plate Phase contrast Thin-film Zernike phase contrast Hilbert differential contrast Cryo-fixation 



We owe the development and biological applications of phase contrast TEM with phase plates to the collaborators as follows. Development: Radostin Danev, Shozo Sugitani, Hiroshi Okawara, Toshiyuki Itoh, Toshikazu Honda, Toshiaki Suzuki, Yoshihiro Arai, Fumio Hosokawa, Sohei Motoki, Rasmus Schroeder and Michael Marko. Applications: Yasuko Kaneko, Koji Nitta, Hitoshi Nakamoto, Nobutaru Usuda, Kimie Atsuzawa, Ayami Nakazawa, Kiyokazu Kametani, Masashi Yamaguchi and Mitsutoshi Setou. This work was supported in part by a Grant-in-aid for Creative Scientific Research from MEXT, Japan and by Core Research for Evolutional Science and Technology (CREST) from JST, Japan.


  1. Aharonov, Y. and Bohm, D. (1959) Significance of electromagnetic potentials in the quantum theory. Phys. Rev. 115: 485–491.CrossRefGoogle Scholar
  2. Alloyeau, D., Hsieh, W.K., Anderson, E.H., Hilken, L., Benner, G., Meng, X., Chen, F.R. and Kisielowski, C. (2010) Imaging of soft and hard materials using a Boersh phase plate in a transmission electron microscope. Ultramicroscopy 110: 563–570.CrossRefGoogle Scholar
  3. Barton, B., Joos, F. and Schröder, R.R. (2008) Improved specimen reconstruction by Hilbert phase contrast tomography. J. Struct. Biol. 164: 210–220.PubMedCrossRefGoogle Scholar
  4. Boersch, H. (1947) Über die Kontraste von Atomen in Electronenmikroskop. Z Naturforschg 2a: 615–633.Google Scholar
  5. Cambie, R., Downing, K.H., Typke, D., Glaeser, R.M. and Jin, J. (2007) Design of a microfabricated, two-electrode phase-contrast element suitable for electron microscopy. Ultramicroscopy 107: 329–339.PubMedCrossRefGoogle Scholar
  6. Danev, R. and Nagayama, K. (2001) Transmission electron microscopy with Zernike phase plate. Ultramicroscopy 88: 243–252.PubMedCrossRefGoogle Scholar
  7. Danev, R., Okawara, H., Usuda, N., Kametani, K. and Nagayama, K. (2002) A novel phase-contrast transmission electron microscopy producing high-contrast topographic images of weak objects. J. Biol. Phys. 28: 627–635.CrossRefGoogle Scholar
  8. Danev, R. and Nagayama, K. (2004) Complex observation in electron microscopy. IV. Reconstruction of complex object wave from conventional and half plane phase plate image pair. J. Phys. Soc. Jpn. 73: 2718–2724.CrossRefGoogle Scholar
  9. Danev, R. and Nagayama, K. (2006) Applicability of thin film phase plates in biological electron microscopy. Biophysics 2: 35–43.CrossRefGoogle Scholar
  10. Danev, R. and Nagayama, K. (2008) Single particle analysis based on Zernike phase contrast transmission electron microscopy. J. Struct. Biol. 161: 211–218.PubMedCrossRefGoogle Scholar
  11. Danev, R. and Nagayama, K. (2010) Phase plates for transmission electron microscopy. Meth. Enzymol. 481: 343–369.Google Scholar
  12. Danev, R., Kanamaru, S., Marko, M. and Nagayama, K. (2010) Zernike phase contrast cryo-electron tomography. J. Struct. Biol. 171(2): 174–181.Google Scholar
  13. Fukuda, Y., Fukazawa, Y., Danev, R., Shigemoto, R. and Nagayama, K. (2009) Tuning of the Zernike phase plate for visualization of detailed ultrastructure in complex biological specimens. J. Struct. Biol. 168: 476–484.PubMedCrossRefGoogle Scholar
  14. Hosogi, N., Shigematsu, H., Terashima, H., Honma, M. and Nagayama, K. (2011) Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from vibrio alginolytius. J. Struct. Biol. 173(1): 67–76.Google Scholar
  15. Huang, S.H., Wang, W.J., Chang, C.S., Hwu, Y.K., Kai, J.J. and Chen, F.R. (2006) The fabrication and application of Zernike electrostatic phase plate. J. Electron Mirosc. 55: 273–280.CrossRefGoogle Scholar
  16. Kaneko, Y., Danev, R., Nitta, K. and Nagayama, K. (2005) In vivo subcellular ultrastructures recognized with Hilbert-differential-contrast transmission electron microscopy. J. Electron Microsc. 54: 79–84.CrossRefGoogle Scholar
  17. Kaneko, Y., Nitta, K. and Nagayama, K. (2007) Observation of in vivo DNA in ice embedded whole cyanobacterial cells by Hilbert differential contrast transmission electron microscopy (HDC-TEM). Plasma Fusion Res. 54: 79–85.Google Scholar
  18. Majorovits, E., Barton, B., Schultheiß, K., Perez-Willard, F., Gerthsen, D. and Schröder R.R. (2007) Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate. Ultramicroscopy 107: 213–226.PubMedCrossRefGoogle Scholar
  19. Matumoto, T. and Tonomura, A. (1996) The phase constancy of electron waves travelling through Boersch’s electrostatic phase plate. Ultramicroscopy 63: 5–10.CrossRefGoogle Scholar
  20. Nagayama, K. (1999) Complex observation in electron microscopy. I. Basic scheme to surpass the Sherzer limit. J. Phys. Soc. Jpn. 68: 811–822.CrossRefGoogle Scholar
  21. Nagayama, K. (2005) Phase contrast enhancement with phase plates in electron microscopy. Ad. Imaging Electr. Phys. 138: 69–146.CrossRefGoogle Scholar
  22. Nagayama, K. (2008) Development of phase plates for electron microscopes and their biological application. Eur. Biophys. J. 37: 345–358.PubMedCrossRefGoogle Scholar
  23. Nitta, K., Nagayama, K., Danev, R. and Kaneko, Y. (2009) Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy. 234: 118–123.Google Scholar
  24. Nomarski, G. (1952) Interferométre á polarization. French Patent 1: 059-123.Google Scholar
  25. Scherzer, O. (1949) The theoretical resolution limit of the electron microscope. J. Appl. Phys. 20: 20–29.CrossRefGoogle Scholar
  26. Shigematsu, H., Sokabe, T., Danev, R., Tominaga, M. and Nagayama, K. (2010) A 3.5-nm structure of rat TRPV4 cation channel revealed by Zernike phase-contrast cryo-EM. J. Biol. Chem. 285: 11210–11218.PubMedCrossRefGoogle Scholar
  27. Shiue, J., Chang, C.S., Huang, S.H., Hsu, C.H., Tsai, J.S., Chang, W.H., Wu, Y.M., Lin, Y.C., Kuo, P.C., Huang, Y.S., Hwu, Y., Kai, J.J., T. F.G. and Chen, F.R. (2009) Phase TEM for biological imaging utilizing a Boersh electrostatic phase plate: theory and practice. J. Electron Microsc. 58: 137–145.Google Scholar
  28. Smith, F.H. (1947) Microscopes. British Patent 639 014, Class 97(i) CroupXX.Google Scholar
  29. Zernike, F. (1942) Phase contrast, a new method for the microscopic observation of transparent objects. Physica 9: 686–698, 974–986.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.National Institute for Physiological SciencesOkazaki CityJapan
  2. 2.The Graduate University for Advanced Studies, School of Physiological SciencesHayamachoJapan

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