New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Envelope structure of semliki forest virus reconstructed from projections of randomly oriented single particles visualized by cryo-electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 174-175
Author(s):  
R.H. Vogel ◽  
S.W. Provencher ◽  
C.-H. von Bonsdorff ◽  
M. Adrian ◽  
J. Dubochet
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Semliki Forest Virus ◽  
Atomic Resolution ◽  
Single Particles ◽  
Basic Principles ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Microscopy Method ◽  
Electron Microscopy Method

The basic principles of the architecture of many viral protein shells are now well established and the structure of several viruses have been elucidated to atomic resolution. Enveloped viruses, like Semliki Forest virus (SFV), have been more difficult to study because they resist crystallization and are easily deformed when prepared for electron microscopy. The latter limitation has been coped with by using a cryo-electron microscopy method in which unfixed and unstained viruses are observed in an unsupported thin layer of vitrified suspension. The appearance of the virus depends strongly on the focus (Fig. 1), because, unlike in conventionally stained specimens, the contrast is essentially due to phase contrast. The focus values of the four micrographs in the series, which were used for the reconstruction, have been chosen to give an optimal coverage of the information contained in the specimen (Fig. 2).

Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex

2013 ◽  
Vol 46 (49) ◽  
pp. 494008 ◽  
Author(s):  
Yi-Min Wu ◽  
Chun-Hsiung Wang ◽  
Jen-wei Chang ◽  
Yi-yun Chen ◽  
Naoyuki Miyazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Protein Complex ◽  
Phase Contrast ◽  
Cryo Electron Microscopy

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

Fourier spectrum analysis of phase contrast of support film

1978 ◽  
Vol 36 (1) ◽  
pp. 170-171
Author(s):  
Tetsuo Oikawa ◽  
Fumiko Ishigaki ◽  
Kiichi Hojou ◽  
Koichi Kanaya
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Phase Shift ◽  
Phase Contrast ◽  
Fourier Transforms ◽  
Spherical Aberration ◽  
High Resolution Electron Microscopy ◽  
Spatial Frequencies ◽  
Resolution Electron ◽  
Atomic Phase

In high resolution electron microscopy, it is most important to determine the defocus of electron micrographs of amorphous support films. The variation of spatial frequencies of phase contrast of support films was obtained from the phase shift of the electron waves caused by defocus and spherical aberration as well as the atomic phase, which are demonstrated by use of optical Fourier transforms. The spatial frequencies of phase contrast of films of tungsten, prepared by ion bombardment, which are useful as support films for high resolution electron microscopy, has been discussed analytically.Taking account of atomic phase shift, the transfer function, which was originally presented by Thon (1966), was modified. Optical Fourier transforms are in similar to the calculated Fourier transforms of corre- sponding computed images. Accordingly, it turned out that the atomic phase shift should not be neglected. The thickness of tungsten film, in case of less than 2 nm thickness, can be determined by comparing the optical Fourier transforms with the calculated ones.

Quantification of micromagnetic structure from Lorentz micrographs

1994 ◽  
Vol 52 ◽  
pp. 894-895
Author(s):  
J. Zweck ◽  
M. Herrmann ◽  
H. Hoffmann
Keyword(s):  
Phase Shift ◽  
Quantitative Evaluation ◽  
Magnetic Induction ◽  
Phase Contrast ◽  
Magnetic Domain ◽  
Contrast Transfer Function ◽  
Lorentz Microscopy ◽  
Domain Structures ◽  
Spatial Frequencies ◽  
Microscopy Images

Defocused imaging of magnetic domain structures is a well-known technique to observe the micromagnetic structures in ferromagnetic thin films. Nevertheless, Lorentz microscopy images are rarely subject to a quantitative evaluation of micromagnetic parameters. In this paper, we offer a new method for quantitative evaluation of ripple wavelengths and ripple angle from Lorentz microscopy images carried out on soft magnetic Ni81Fe19 films. The work was carried out using a Philips CM30 electron microscope with a combined Twin/Lorentz lens.The experiments were performed on thin ferromagnetic films of a Ni81Fe19 alloy. Due to the internal magnetic induction within the specimens, the partial electron waves experience a phase shift proportional to the local in-plane magnetic induction , the specimen's thickness t and the lateral distance x from an arbitrarily chosen point of zero phase shift on the specimen. This phase shift can then be imaged using phase contrast methods similar to HREM. Since the phase shifts and the corresponding deflection angles can be very small, a large defocus is necessary to obtain contrast. This large defocus gives rise to an oscillating phase contrast transfer function for the spatial frequencies under observation as well as to a damping envelope for higher spatial frequencies.

Zernike Phase Contrast Cryo-Electron Microscopy of Virus Particles

2012 ◽  
Vol 18 (S2) ◽  
pp. 462-463
Author(s):  
W. Chiu ◽  
X. Liu ◽  
K. Murata ◽  
H. Khant ◽  
R.H. Rochat ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Virus Particles ◽  
Cryo Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

An Improved Holey Carbon Film for Cryo-Electron Microscopy

2007 ◽  
Vol 13 (5) ◽  
pp. 365-371 ◽  
Author(s):  
Joel Quispe ◽  
John Damiano ◽  
Stephen E. Mick ◽  
David P. Nackashi ◽  
Denis Fellmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Particle Distribution ◽  
Carbon Film ◽  
Soft Materials ◽  
Carbon Films ◽  
Water Soluble ◽  
Specimen Preparation ◽  
Dramatic Improvement ◽  
Single Particles ◽  
Cryo Electron Microscopy

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

scholarly journals A WAVELET BASED ALTERNATIVE ITERATION METHOD FOR THE ORIENTATION REFINEMENT OF CRYO-ELECTRON MICROSCOPY 3D RECONSTRUCTION

2015 ◽  
Vol 20 (3) ◽  
pp. 396-408 ◽  
Author(s):  
Zhucui Jing ◽  
Ming Li
Keyword(s):  
Electron Microscopy ◽  
Iteration Method ◽  
Three Dimensional ◽  
Particle Method ◽  
Biological Macromolecules ◽  
Orthonormal Bases ◽  
Cryo Electron Microscopy ◽  
Marquardt Algorithm ◽  
Density Map ◽  
Particle Images

Cryo-electron microscopy (cryo-EM) single particle method (SPM) reconstructs the three-dimensional (3D) density map of biological macromolecules using 2D particle images with estimated orientations. The estimated orientations have errors which result in the decrease in resolution of the reconstructed map. We propose a wavelet orthonormal bases based iteration method by refining alternatively the orientations and the map using Levenberg–Marquardt algorithm and soft-thresholding, respectively. The convergence analysis of the proposed algorithm is provided and numerical experiments for simulated particle images show its good performance.

scholarly journals A microtubule RELION-based pipeline for cryo-EM image processing

2019 ◽  
Author(s):  
Alexander D. Cook ◽  
Szymon W. Manka ◽  
Su Wang ◽  
Carolyn A. Moores ◽  
Joseph Atherton
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Structural Similarity ◽  
Geometric Lattice ◽  
Contrast Transfer Function ◽  
Biologically Relevant ◽  
Cryo Electron Microscopy

AbstractMicrotubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.AbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

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