Spectral DQE of the Volta Phase Plate

ABSTRACTThe Volta Phase Plate (VPP) consists of a heated, thin film that is placed in the same plane as the focused diffraction pattern of an electron microscope. A change in surface potential develops at the point irradiated by the intense, unscattered electron beam, and this altered surface potential produces, in turn, a phase shift between the unscattered and scattered parts of the electron wave. While the VPP thus increases the image contrast for weak-phase objects at low spatial frequencies, we report here that it also leads to the loss of an increasing fraction of the signal at higher resolution. The approximately linear dependence (with increasing resolution) of this loss has been quantified at 200 kV and 300 kV, using evaporated-carbon films of different thicknesses as Volta phase plates. In all cases, the loss of signal remains almost independent of variation of the conditions and parameters that were tested. In spite of having done a number or additional, discovery-based experiments, the cause of this loss of signal remains unexplained at this point.

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Phase Contrast Enhancement with Phase Plates in Biological Electron Microscopy

Microscopy Today ◽

10.1017/s1551929510000465 ◽

2010 ◽

Vol 18 (4) ◽

pp. 10-13 ◽

Cited By ~ 1

Author(s):

Kuniaki Nagayama ◽

Radostin Danev ◽

Hideki Shigematsu ◽

Naoki Hosogi ◽

Yoshiyuki Fukuda ◽

...

Keyword(s):

Thin Film ◽

Electron Microscopy ◽

Phase Contrast ◽

Phase Plate ◽

Electron Loss ◽

Whole Cells ◽

Transmission Electron ◽

Different Types ◽

Transparent Objects ◽

Phase Plates

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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Structure of Freeze-Fractured, Deep-Etched F-Actin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098617 ◽

1981 ◽

Vol 39 ◽

pp. 422-423

Author(s):

G.C. Ruben ◽

N.S. Allen ◽

J.L. Travis

Keyword(s):

Thin Film ◽

Electron Beam ◽

Quartz Crystal ◽

Carbon Films ◽

Uranyl Acetate ◽

Electron Microscopic ◽

Resolution Electron ◽

Current Setting ◽

Stereoscopic Visualization ◽

A Current

High resolution electron microscopic images of F-actin, which had been rapidly frozen, freeze-fractured and deep-etched, have been compared with conventional negatively stained images. Our methods permit the stereoscopic visualization of native actin filament substructureF-actin, prepared by the method of Spudich and Watt (1) was dialized against 2 mM MgCl2 and 5 mM Tris at pH 7. F-actin was then negatively stained (1% uranyl acetate at pH 4.5) on 10 nm carbon films (2) or frozen on Balzer's gold pedestals in liquid propane at -190°C. After placing the specimen in a Balzer's 300 freezing microtome, both electron beam guns were out-gassed for two minutes at a current setting of 5. During this process the knife arm (-196°C) covered the sample. The specimen was freeze-etched for 3 minutes at -97°C under the knife arm. Then the sample stage was cooled (-120°C) for two minutes (5xl0-7 torr) before the Pt-C was evaporated at a 45° angle to a preset thickness of 2.5 and 2.9 nm on a QSG quartz crystal thin film monitor.

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A Study of Electrostatic Phase Plates using Electron Holography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100164805 ◽

1996 ◽

Vol 54 ◽

pp. 468-469

Author(s):

A. Mohan ◽

B. G. Frost ◽

D. C. Joy

Keyword(s):

Electron Beam ◽

Scattered Wave ◽

Phase Shifting ◽

Objective Lens ◽

Phase Plate ◽

Electron Holography ◽

Small Deviations ◽

Transmission Electron ◽

Contrast Microscopy ◽

Phase Plates

Contrast in transmission electron microscopy is normally provided by variations in the intensity of the transmitted electron beam, but weakly scattering objects such as biological tissue and polymers may not cause a sufficient change in amplitude to provide a useful image. The phase of the electron beam, however, is altered and if the phase change can be made to yield amplitude variation then the imaging can be carried out as desired. This can be accomplished by the use of some phase shifting device which alters the phase of the scattered wave by π/2 with respect to the unscattered wave. A number of such devices have been discussed in the literature but their design has typically been optimized empirically. One of the methods for performing Phase Contrast Microscopy in the TEM is by using an electrostatic phase plate in the back focal plane of the objective lens. This device consists of a charged fiber which causes small deviations in the ray paths of the scattered and the unscattered waves thus introducing a suitable phase shift between them.

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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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Charging of Thin Film Phase Plates under Electron Beam Irradiation

Microscopy and Microanalysis ◽

10.1017/s1431927614002876 ◽

2014 ◽

Vol 20 (S3) ◽

pp. 230-231 ◽

Cited By ~ 2

Author(s):

Marek Malac ◽

Marco Beleggia ◽

Ray Egerton ◽

Masahiro Kawasaki ◽

Michael Berge ◽

...

Keyword(s):

Thin Film ◽

Electron Beam ◽

Electron Beam Irradiation ◽

Beam Irradiation ◽

Phase Plates

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Thin-Film Phase Plates for Transmission Electron Microscopy Fabricated from Metallic Glasses

Microscopy and Microanalysis ◽

10.1017/s143192761601165x ◽

2016 ◽

Vol 22 (5) ◽

pp. 955-963 ◽

Cited By ~ 8

Author(s):

Manuel Dries ◽

Simon Hettler ◽

Tina Schulze ◽

Winfried Send ◽

Erich Müller ◽

...

Keyword(s):

Thin Film ◽

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Amorphous Carbon ◽

Mean Free Path ◽

Amorphous Structure ◽

Small Probability ◽

Spatial Frequencies ◽

Transmission Electron ◽

Phase Plates

AbstractThin-film phase plates (PPs) have become an interesting tool to enhance the contrast of weak-phase objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon, which suffers from quick degeneration under the intense electron-beam illumination. Recent investigations have focused on the search for alternative materials with an improved material stability. This work presents thin-film PPs fabricated from metallic glass alloys, which are characterized by a high electrical conductivity and an amorphous structure. Thin films of the zirconium-based alloy Zr65.0Al7.5Cu27.5(ZAC) were fabricated and their phase-shifting properties were evaluated. The ZAC film was investigated by different TEM techniques, which reveal beneficial properties compared with amorphous carbon PPs. Particularly favorable is the small probability for inelastic plasmon scattering, which results from the combined effect of a moderate inelastic mean free path and a reduced film thickness due to a high mean inner potential. Small probability plasmon scattering improves contrast transfer at high spatial frequencies, which makes the ZAC alloy a promising material for PP fabrication.

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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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Object Wave Determination by Single-Sideband Methods

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100071776 ◽

1973 ◽

Vol 31 ◽

pp. 266-267

Author(s):

Kenneth H. Downing ◽

Benjamin M. Siegel

Keyword(s):

Phase Shift ◽

Carbon Films ◽

Object Wave ◽

Electron Wave ◽

Phase Object ◽

Heavy Atoms ◽

Single Sideband ◽

Thin Carbon ◽

Additional Phase Shift ◽

Additional Phase

Under the “weak phase object” approximation, the component of the electron wave scattered by an object is phase shifted by π/2 with respect to the unscattered component. This phase shift has been confirmed for thin carbon films by many experiments dealing with image contrast and the contrast transfer theory. There is also an additional phase shift which is a function of the atomic number of the scattering atom. This shift is negligible for light atoms such as carbon, but becomes significant for heavy atoms as used for stains for biological specimens. The light elements are imaged as phase objects, while those atoms scattering with a larger phase shift may be imaged as amplitude objects. There is a great deal of interest in determining the complete object wave, i.e., both the phase and amplitude components of the electron wave leaving the object.

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Characteristics of an Electrostatic Phase Plate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100096345 ◽

1972 ◽

Vol 30 ◽

pp. 618-619

Author(s):

William Krakow ◽

Benjamin Siegel

Keyword(s):

Phase Contrast ◽

Objective Lens ◽

Phase Plate ◽

Net Charge ◽

Phase Plates ◽

Beam Currents ◽

Contrast Image ◽

Bright Phase ◽

Additional Phase Shift ◽

Additional Phase

Unwin has used a metallized non-conducting thread in the back focal plane of the objective lens that stops out a portion of the unscattered beam, takes on a localized positive charge and thus produces an additional phase shift to give a different transfer function of the lens. Under the particular conditions Unwin used, the phase contrast image was shifted to bright phase contrast for optimum focus.We have investigated the characteristics of this type of electrostatic phase plate, both analytically and experimentally, as functions of the magnitude of charge and defocus. Phase plates have been constructed by using Wollaston wire to mount 0.25μ diameter platinum wires across apertures ranging from 50 to 200μ diameter and vapor depositing SiO and gold on the mounted wires to give them the desired charging characteristics. The net charge was varied by adjusting only the bias on the Wehnelt shield of the gun, and hence the beam currents and effective size of the source.

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