Which is Better for Protein Imaging: Phase Contrast TEM or Annular Dark Field STEM?

In a landmark paper Henderson compared X-ray, neutrons and electrons for protein structure determination. He showed that electron microscopy should be superior to X-ray or neutron diffraction in terms of dose for a given resolution. in addition he presented a theoretical analysis to determine the smallest size molecule whose structure could be determined by phase contrast microscopy. Although he qualitatively considered amplitude contrast mechanisms and concluded they were inferior to phase contrast, no explicit numerical analysis was performed. It has been implicitly assumed that bright field phase contrast imaging is the optimal technique for imaging small biological molecules. Protein specimens are usually embedded in some medium such as ice or glucose. Since they must give a very low contrast it seems reasonable to expect that bright field techniques for these weakly scattering objects would be inferior, given that a weak signal is sitting on large background.

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Removal of inelastically scattered electrons substantially increases phase contrast on frozen-hydrated molecules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100127694 ◽

1987 ◽

Vol 45 ◽

pp. 652-653

Author(s):

John P. Langmore ◽

Brian D. Athey

Keyword(s):

Electron Diffraction ◽

Phase Contrast ◽

Low Dose ◽

Dark Field ◽

Biological Structure ◽

Bright Field ◽

Low Contrast ◽

Negative Stain ◽

The Difference ◽

Better Than

Although electron diffraction indicates better than 0.3nm preservation of biological structure in vitreous ice, the imaging of molecules in ice is limited by low contrast. Thus, low-dose images of frozen-hydrated molecules have significantly more noise than images of air-dried or negatively-stained molecules. We have addressed the question of the origins of this loss of contrast. One unavoidable effect is the reduction in scattering contrast between a molecule and the background. In effect, the difference in scattering power between a molecule and its background is 2-5 times less in a layer of ice than in vacuum or negative stain. A second, previously unrecognized, effect is the large, incoherent background of inelastic scattering from the ice. This background reduces both scattering and phase contrast by an additional factor of about 3, as shown in this paper. We have used energy filtration on the Zeiss EM902 in order to eliminate this second effect, and also increase scattering contrast in bright-field and dark-field.

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A proof-of principal study using phase-contrast imaging for the detection of large airway pathologies after lung transplantation

Scientific Reports ◽

10.1038/s41598-020-75185-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Stephan Umkehrer ◽

Carmela Morrone ◽

Julien Dinkel ◽

Laura Aigner ◽

Maximilian F. Reiser ◽

...

Keyword(s):

Lung Transplantation ◽

Phase Contrast ◽

Small Animal ◽

Lung Parenchyma ◽

Dark Field ◽

Contrast Imaging ◽

X Ray ◽

Contrast Mechanism ◽

Contrast Information

Abstract In this study we aim to evaluate the assessment of bronchial pathologies in a murine model of lung transplantation with grating-based X-ray interferometry in vivo. Imaging was performed using a dedicated grating-based small-animal X-ray dark-field and phase-contrast scanner. While the contrast modality of the dark-field signal already showed several promising applications for diagnosing various types of pulmonary diseases, the phase-shifting contrast mechanism of the phase contrast has not yet been evaluated in vivo. For this purpose, qualitative analysis of phase-contrast images was performed and revealed pathologies due to previous lung transplantation, such as unilateral bronchial stenosis or bronchial truncation. Dependent lung parenchyma showed a strong loss in dark-field and absorption signal intensity, possibly caused by several post transplantational pathologies such as atelectasis, pleural effusion, or pulmonary infiltrates. With this study, we are able to show that bronchial pathologies can be visualized in vivo using conventional X-ray imaging when phase-contrast information is analysed. Absorption and dark-field images can be used to quantify the severity of lack of ventilation in the affected lung.

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Detection of individual sub-pixel features in edge-illumination x-ray phase contrast imaging by means of the dark-field channel

Journal of Physics D Applied Physics ◽

10.1088/1361-6463/ab5aa0 ◽

2019 ◽

Vol 53 (9) ◽

pp. 095401

Author(s):

Norihito Matsunaga ◽

Kazuhiro Yano ◽

Marco Endrizzi ◽

Alessandro Olivo

Keyword(s):

Phase Contrast ◽

Dark Field ◽

Phase Contrast Imaging ◽

Contrast Imaging ◽

X Ray

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Factors Affecting the Spatial Resolution in 2D Grating–Based X-Ray Phase Contrast Imaging

Frontiers in Physics ◽

10.3389/fphy.2021.672207 ◽

2021 ◽

Vol 9 ◽

Author(s):

Siwei Tao ◽

Congxiao He ◽

Xiang Hao ◽

Cuifang Kuang ◽

Xu Liu

Keyword(s):

Spatial Resolution ◽

Phase Contrast ◽

Imaging System ◽

Diffraction Theory ◽

Dark Field ◽

Phase Contrast Imaging ◽

Contrast Imaging ◽

Transform Method ◽

Factors Affecting ◽

X Ray

X-ray phase contrast imaging is a promising technique in X-ray biological microscopy, as it improves the contrast of images for materials with low electron density compared to traditional X-ray imaging. The spatial resolution is an important parameter to evaluate the image quality. In this paper, simulation of factors which may affect the spatial resolution in a typical 2D grating–based phase contrast imaging system is conducted. This simulation is based on scalar diffraction theory and the operator theory of imaging. Absorption, differential phase contrast, and dark-field images are retrieved via the Fourier transform method. Furthermore, the limitation of the grating-to-detector distance in the spatial harmonic method is discussed in detail.

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Calculations of Z-Contrast for organic and biological specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007521x ◽

1983 ◽

Vol 41 ◽

pp. 284-285

Author(s):

R.F. Egerton ◽

M. Misra

Keyword(s):

Atomic Number ◽

Cross Sections ◽

Phase Contrast ◽

Detection System ◽

Dark Field ◽

Single Atom ◽

Bright Field ◽

Annular Dark Field ◽

Z Contrast ◽

Increasing Function

So-called "atomic-number contrast" is obtained in STEM by displaying a ratio signal formed by dividing the annular-dark-field signal Iad by the inelastic component Ii of the bright-field intensity (isolated by means of an electron spectrometer; see Fig. 1). Originally used for single-atom imaging, the technique has more recently been applied to polymer samples and biological tissue.We report here estimates of the ratio signal from organic specimens, based on the following assumptions:(1) That the specimen is amorphous and that phase contrast may be neglected for the electron-optical conditions and specimen features being considered; (2) That atomic cross sections may be used to estimate the amount of elastic and inelastic scattering. Modern calculations differ from simple Lenz theory in predicting that the cross section is not a smoothly-increasing function of atomic number (see Fig. 2), particularly for the 1ighter elements. (3) We assume a slightly idealized detection system in which all elastically scattered electrons contribute to Iad, while all electrons which have been inelastically (but not elastically) scattered contribute to Ii.

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X-ray dark-field and phase-contrast imaging using a grating interferometer

Journal of Applied Physics ◽

10.1063/1.3115639 ◽

2009 ◽

Vol 105 (10) ◽

pp. 102006 ◽

Cited By ~ 53

Author(s):

F. Pfeiffer ◽

M. Bech ◽

O. Bunk ◽

T. Donath ◽

B. Henrich ◽

...

Keyword(s):

Phase Contrast ◽

Dark Field ◽

Phase Contrast Imaging ◽

Contrast Imaging ◽

X Ray ◽

Grating Interferometer

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Phase-Contrast and Dark-Field Imaging

Journal of Imaging ◽

10.3390/jimaging4100113 ◽

2018 ◽

Vol 4 (10) ◽

pp. 113

Author(s):

Simon Zabler

Keyword(s):

Phase Contrast ◽

Dark Field ◽

X Rays ◽

Reconstruction Algorithms ◽

Contrast Imaging ◽

Full Field ◽

X Ray ◽

Dark Field Imaging ◽

Contrast Mechanisms ◽

Field Imaging

Very early, in 1896, Wilhelm Conrad Röntgen, the founding father of X-rays, attempted to measure diffraction and refraction by this new kind of radiation, in vain. Only 70 years later, these effects were measured by Ulrich Bonse and Michael Hart who used them to make full-field images of biological specimen, coining the term phase-contrast imaging. Yet, another 30 years passed until the Talbot effect was rediscovered for X-radiation, giving rise to a micrograting based interferometer, replacing the Bonse–Hart interferometer, which relied on a set of four Laue-crystals for beam splitting and interference. By merging the Lau-interferometer with this Talbot-interferometer, another ten years later, measuring X-ray refraction and X-ray scattering full-field and in cm-sized objects (as Röntgen had attempted 110 years earlier) became feasible in every X-ray laboratory around the world. Today, now that another twelve years have passed and we are approaching the 125th jubilee of Röntgen’s discovery, neither Laue-crystals nor microgratings are a necessity for sensing refraction and scattering by X-rays. Cardboard, steel wool, and sandpaper are sufficient for extracting these contrasts from transmission images, using the latest image reconstruction algorithms. This advancement and the ever rising number of applications for phase-contrast and dark-field imaging prove to what degree our understanding of imaging physics as well as signal processing have advanced since the advent of X-ray physics, in particular during the past two decades. The discovery of the electron, as well as the development of electron imaging technology, has accompanied X-ray physics closely along its path, both modalities exploring the applications of new dark-field contrast mechanisms these days. Materials science, life science, archeology, non-destructive testing, and medicine are the key faculties which have already integrated these new imaging devices, using their contrast mechanisms in full. This special issue “Phase-Contrast and Dark-field Imaging” gives us a broad yet very to-the-point glimpse of research and development which are currently taking place in this very active field. We find reviews, applications reports, and methodological papers of very high quality from various groups, most of which operate X-ray scanners which comprise these new imaging modalities.

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Quantitative phase retrieval in X-ray Zernike phase contrast microscopy

Journal of Synchrotron Radiation ◽

10.1107/s1600577515007699 ◽

2015 ◽

Vol 22 (4) ◽

pp. 1056-1061 ◽

Cited By ~ 4

Author(s):

Heng Chen ◽

Zhili Wang ◽

Kun Gao ◽

Qiyue Hou ◽

Dajiang Wang ◽

...

Keyword(s):

Phase Contrast ◽

Phase Retrieval ◽

Materials Science ◽

Phase Contrast Microscopy ◽

Contrast Imaging ◽

X Ray ◽

Quantitative Phase ◽

Direct Light ◽

Contrast Microscopy ◽

Phase Variations

In recent years, increasing attention has been devoted to X-ray phase contrast imaging, since it can provide high-contrast images by using phase variations. Among the different existing techniques, Zernike phase contrast microscopy is one of the most popular phase-sensitive techniques for investigating the fine structure of the sample at high spatial resolution. In X-ray Zernike phase contrast microscopy, the image contrast is indeed a mixture of absorption and phase contrast. Therefore, this technique just provides qualitative information on the object, which makes the interpretation of the image difficult. In this contribution, an approach is proposed for quantitative phase retrieval in X-ray Zernike phase contrast microscopy. By shifting the phase of the direct light by π/2 and 3π/2, two images of the same object are measured successively. The phase information of the object can then be quantitatively retrieved by a proper combination of the measured images. Numerical experiments were carried out and the results confirmed the feasibility of the proposed method. It is expected that the proposed method will find widespread applications in biology, materials science and so on.

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Imaging of Silicon (111) at 1.92Å Resolution using a 100keV STEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178926 ◽

1990 ◽

Vol 48 (1) ◽

pp. 34-35

Author(s):

Peirong Xu

Keyword(s):

High Resolution ◽

Phase Contrast ◽

Threshold Energy ◽

Dark Field ◽

Observation Time ◽

Operating Voltage ◽

Pole Piece ◽

Field Phase ◽

Bright Field ◽

Annular Dark Field

Atomic structure imaging using bright field phase contrast at less than 2Å resolution has become routinely possible in medium and high voltage microscopes (>200 keV). Radiation damage at these elevated voltages can be serious and this limits the length of useful observation time. For example, the knock-on threshold energy for silicon is 120-190keV. Recently, a VG HB501A STEM equipped with a newly developed ultra-high resolution pole piece (Cs=0.7mm) has demonstrated the capability of achieving sub-2Å resolution in imaging the (111) silicon latticer using both bright field (BF) and annular dark field (ADF) modes at an operating voltage of l00keV (Fig.1).A thin silicon specimen was prepared through successive steps of chemical etching, anodic etching and reactive ion etching. Large flat thin areas about 100Å thick were produced in the specimen. Since there is no tilting mechanism for the stage used with this ultra-high resolution pole piece, the specimen was not oriented exactly along the (111) zone axis as indicated by CBED but was less than 1-2° off.

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