Phase Contrast Enhancement with Phase Plates in Biological Electron Microscopy

Theoretically, transmission electron microscopy (TEM) is compatible with three different types of phase plate: thin-film, electrostatic, and magnetic. However, designing functional phase plates has been an arduous process that has suffered from unavoidable technical obstacles such as phase-plate charging and difficulties associated with micro-fabrication of electrostatic and magnetic phase plates. This review discusses phase-contrast schemes that allow visualization of transparent objects with high contrast. Next it deals with recent studies on biological applications ranging from proteins and viruses to whole cells. Finally, future prospects for overcoming the problem of phase-plate charging and for designing the next generation of phase-plates to solve the problem of electron loss inherent in thin-film phase plates are discussed.

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Phase contrast electron microscopy: development of thin-film phase plates and biological applications

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2008.2268 ◽

2008 ◽

Vol 363 (1500) ◽

pp. 2153-2162 ◽

Cited By ~ 48

Author(s):

Kuniaki Nagayama ◽

Radostin Danev

Keyword(s):

Thin Film ◽

Electron Microscopy ◽

Amorphous Carbon ◽

Phase Contrast ◽

Carbon Film ◽

Objective Lens ◽

Phase Plate ◽

Amorphous Carbon Film ◽

Transmission Electron ◽

Phase Plates

Phase contrast transmission electron microscopy (TEM) based on thin-film phase plates has been developed and applied to biological systems. Currently, development is focused on two techniques that employ two different types of phase plates. The first technique uses a Zernike phase plate, which is made of a uniform amorphous carbon film that completely covers the aperture of an objective lens and can retard the phase of electron waves by π /2, except at the centre where a tiny hole is drilled. The other technique uses a Hilbert phase plate, which is made of an amorphous carbon film that is twice as thick as the Zernike phase plate, covers only half of the aperture and retards the electron wave phase by π . By combining the power of efficient phase contrast detection with the accurate preservation achieved by a cryotechnique such as vitrification, macromolecular complexes and supermolecular structures inside intact bacterial or eukaryotic cells may be visualized without staining. Phase contrast cryo-TEM has the potential to bridge the gap between cellular and molecular biology in terms of high-resolution visualization. Examples using proteins, viruses, cyanobacteria and somatic cells are provided.

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

Microscopy and Microanalysis ◽

10.1017/s1431927613013901 ◽

2014 ◽

Vol 20 (1) ◽

pp. 175-183 ◽

Cited By ~ 13

Author(s):

Nicole Frindt ◽

Marco Oster ◽

Simon Hettler ◽

Björn Gamm ◽

Levin Dieterle ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Phase Contrast ◽

Structural Information ◽

Signal To Noise Ratio ◽

Phase Plate ◽

Spatial Frequencies ◽

Transmission Electron ◽

Sample Orientation ◽

Phase Plates

AbstractTransmission 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.

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Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s143192761601196x ◽

2016 ◽

Vol 22 (6) ◽

pp. 1316-1328 ◽

Cited By ~ 7

Author(s):

Michael Marko ◽

Chyongere Hsieh ◽

Eric Leith ◽

David Mastronarde ◽

Sohei Motoki

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Practical Experience ◽

Phase Plate ◽

Native State ◽

Automated Data Collection ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Phase Plates ◽

Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

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