bluuebunny/crossref_metadata_2025_split huggingface dataset:
Information Enhancement in the STEM by Employing a Multiple-Signals Imaging Procedure
Authors: Hammel M. , Kohl H. , Rose H. 
Published: June 2020

When performing high resolution bright field imaging of weak phase objects in a STEM, all electrons falling onto the specimen are reaching the detector plane. Hence, there is no information available about the object function if the detector covers the entire cone of the unscattered electrons. Nevertheless, the current density within this cone differs from being constant because of constructive and destructive interference between the elastically scattered and unscattered electrons. In order to get a phase contrast signal, one can use a circular detector subtending a small aperture angle. However, this method discards a lot of electrons which have been scattered by the specimen and therefore carry information about its structure. In principle, all of them should be subject to investigation. To achieve this, the detector region must be divided in such a way that regions with constructive and destructive interference, respectively, form different signals. Since these signals are registered separately, they can be appropriately combined to extract the information of interest.Several methods have been proposed for optimizing the detector configuration of the multiple signals imaging mode in the last fifteen years. Splitting the detector into semicircles yields differential phase contrast and explores the gradient of the object function. Integrating the signal of such a detector in the direction perpendicular to the split gives good contrast transfer at small spatial frequencies at least at high doses. On the other hand, a division of the circular detector in rings enables one to improve the transfer of high spatial frequencies without any zeroes in the phase contrast transfer function (PCTF, [2]). Both detector arrangements have been combined. Recently, a free configurational detector has been developed and tested. The configuration of ref. [3] has been improved by M. Haider et al. so that the possibilities in creating zones of different shapes have increased very much.
Dynamic Focussing of Tilted Specimens in the CM TEM via Remote Control
Authors: de Jong Marc J.C. , Typke Dieter 
Published: June 2020

The freedom of tilting specimens is essential in many fields of TEM research. Examples in life science include stereomicrography of thick specimens and the reduction of the missing cone information in 3D reconstructions by means of projected images acquired from a specimen tilt series. The difference in focus across the image and the concomitant change in contrast are two serious limitations in using the available tilt flexibility.Instead of illuminating the area of interest with a broad coherent beam as is normally used in conventional TEM imaging, the object could be exposed by a small spot that is being scanned parallel to tilt axis (Fig. 1). In this case the image is acquired on a photographic plate by scanning this beam across the area of interest. After a full line has been scanned, the focus of the imaging system can be adjusted to correct for the height difference in the tilted object and the next line can be imaged with the correct focus conditions onto the photographic plate. An additional benefit of this "DYNAFOCUS" technique is the improvement in image quality at high resolution of radiation sensitive specimens (Fig. 2). This can be explained by the reduction of beam-induced object movements due to the small spot illumination.
Application of Imaging Plate to High-Voltage Electron Microscopy
Authors: Isoda Seiji , Saitoh Kimitsugu , Moriguchi Sakumi , Kobayashi Takashi 
Published: June 2020

On the observation of structures by high resolution electron microscopy, recording materials with high sensitivity and high quality is awaited, especially for the study of radiation sensitive specimens. Such recording material should be easily combined with the minimum dose system and cryoprotection method. Recently a new recording material, imaging plate, comes to be widely used in X-ray radiography and also in electron microscopy, because of its high sensitivity, high quality and the easiness in handling the images with a computer. The properties of the imaging plate in 100 to 400 kV electron microscopes were already discussed and the effectiveness was revealed.It is demanded to study the applicability of the imaging plate to high voltage electron microscopy. The quality of the imaging plate was investigated using an imaging plate system (JEOL EM-HSR100) equipped in a new Kyoto 1000kV electron microscope. In the system both the imaging plate and films can be introduced together into the camera chamber. Figure 1 shows the effect of accelerating voltage on read-out signal intensities from the imaging plate. The characteristic of commercially available imaging plates is unfortunately optimized for 100 to 200 keV electrons and then for 600 to 1000 keV electrons the signal is reduced. In the electron dose range of 10−13 to 10−10 C/cm2, the signal increases linearly with logarithm of electron dose in all acceralating volatges.
Quantification of High Resolution Electron Microscopy and Electron Diffraction by the Imaging Plate
Authors: Shindo Daisuke , Hiraga Kenji , Hirabayashi Makoto , Oikawa Tetsuo , Mori Nobufumi 
Published: June 2020

Taking advantages of “Imaging Plate” (IP), i.e., a wide dynamic range and good linearity for electron intensity, high resolution electron microscope (HREM) images and electron diffraction (ED) patterns were quantitatively investigated. Observations of HREM images and ED patterns were made by the TEM-IP System (PIXsys TEM) using electron microscopes JEM-4000EX and JEM-2000FX, respectively. The data on the IP with an effective size of 102×77 mm2 was converted into digital data of 2048 × 1536 pixels with 4096 gray levels and analyzed with the IP-processor of the PIXsys TEM and a large-computer ACOS-2020.Since the IP is highly sensitive to incident electrons, the beam convergence can be lowered for HREM observations, and so the effect of the dumping envelope due to the beam divergence can be reduced. In Fig. 1, an HREM image of an oxide W-Ta-O, taken at a direct magnification of 1,500,000x and an exposure time of 2 s, shows fine contrast of partially ordered cation distributions. Although the phase contrast of the HREM image is rather weak, the image was recorded with about 1100 effective gray levels which are much larger than the 256 gray levels normally used in a conventional image processing system. Intensity distributions in two parts of the image near the crystal edge (A) and at a relatively thicker region (B) are visualized with perspective drawings in Figs. 2(a) and 2(b), and compared quantitatively with contour maps in Figs. 3(a) and 3(b), respectively. It was noticed that projected atomic potentials of the block structure unit (see the inset of Fig. 1) were revealed faithfully in A, having intensity minima at heavy atomic columns (H), whereas the low potential regions (L) have sharp intensity maxima at the thicker region B. These observed images will be compared quantitatively with calculated ones, taking into account the dynamical diffraction effect.
Electron Trajectories Calculations of an Energy - Filtering Field-Emission Gun
Authors: Troyon Michel , Lei He Ning 
Published: June 2020

In many cases, the contribution of beam energy spread to the limitation of the performances of an electron microscope is strong. In the case of the field emission gun (FEG) , Troyon has experimentally shown it is possible to reduce considerably the energy spread by energy filtering at the gun level. The system developed consists basically of a magnetic FEG with a retarding electrode working as the retarding electrode of an energy filter. The principle is recalled in Fig. 1 and the cross section of the accelerator is given in Fig. 2. In this paper, the results of electron trajectories calculations inside the energy filtering field emission gun (EFFEG) are given.Fig. 3 shows that electrons of same energy, but entering the retarding field with different angles, can have exit angles very different. Due to the work function of approximately 4.5 eV the electrons, for an extracting potential Vo = 2 kV, enter in the field of the retarding electrode with an energy smaller than 2 keV. In Fig. 3 trajectories are computed for an electron of 1996 eV. Electrons passing by the nodal points have the same entering and exit angles. Trajectory 1 in Fig. 3 corresponds to an entering radius re = 17.5 μm and an entering semi angle αe = 1.2 mrad. For these re and αe values, at Vr =6 V, the exit semi angle αs = αe . Fig. 3 shows that an electron entering parallely to the axis, even very close to the axis (re = 10 μm) has a larger exit angle than electrons passing by the nodal points.
Phase Shift of Electron Waves in A Rotating Frame of Reference
Authors: Hasselbach F. , Nicklaus M. 
Published: June 2020

After the first matter wave version of Sagnac’s classical light optical experiment of 1913, performed by Mercereau and Zimmermann with electron Cooper pairs in 1965, and the Sagnac experiment realized with neutrons by Werner et al. in 1979 , we report here on the first observation of the rotational phase shift of electron waves in vacuum.Theory. The Sagnac effect links classical physics, quantum physics and relativity. Using the special theory of relativity it can be derived that coherent waves, e.g. of light, neutrons or electrons, travelling around a finite area A experience a relative phaseshift
Atomic Resolution in Crystal Aperture Stem
Authors: Fourie J T 
Published: June 2020

The attempts at improvement of electron optical systems to date, have largely been directed towards the design aspect of magnetic lenses and towards the establishment of ideal lens combinations. In the present work the emphasis has been placed on the utilization of a unique three-dimensional crystal objective aperture within a standard electron optical system with the aim to reduce the spherical aberration without introducing diffraction effects. A brief summary of this work together with a description of results obtained recently, will be given.The concept of utilizing a crystal as aperture in an electron optical system was introduced by Fourie who employed a {111} crystal foil as a collector aperture, by mounting the sample directly on top of the foil and in intimate contact with the foil. In the present work the sample was mounted on the bottom of the foil so that the crystal would function as an objective or probe forming aperture. The transmission function of such a crystal aperture depends on the thickness, t, and the orientation of the foil. The expression for calculating the transmission function was derived by Hashimoto, Howie and Whelan on the basis of the electron equivalent of the Borrmann anomalous absorption effect in crystals. In Fig. 1 the functions for a g220 diffraction vector and t = 0.53 and 1.0 μm are shown. Here n= Θ‒ΘB, where Θ is the angle between the incident ray and the (hkl) planes, and ΘB is the Bragg angle.
3D Reconstruction of Single Particles by Quasi-Conical Tilting from Micrographs Recorded with Dynamic Focusing
Authors: Typke D. , Pfeifer G. , Hegerl R. , Baumeister W. 
Published: June 2020

The method of quasi-conical tilting for the 3D reconstruction of single macromolecules utilizes the fact that particles tend to adsorb to the supporting film in a few preferred orientations, though with arbitrary azimuthal angles. Therefore, a single electron micrograph of a tilted specimen containing a large number of particle projections provides the data of a conical tilt series with projection directions distributed randomly on a cone. For the selection of particles of equal orientation with respect to the supporting film and for the determination of their azimuth angles, a zero tilt image taken after the minimum dose “tilt image” is utilized.A disadvantage of the method is the large focal range of the tilt image, which e.g. at 30,000× magnification exceeds 5 μm. This, however, can be overcome by small-spot illumination in conjunction with dynamic focusing. For this, the illuminating beam is reduced to a diameter of 50-200 nm, and shifted stepwise or continuously over the object; for each spot position the focus is properly adjusted. Thus the focus range of the whole image is restricted to that of a single spot. Another way to realize dynamic focusing of a tilted object is the use of a narrow slit aperture in the condenser C2, thus illuminating a straight line parallel to the tilt axis.
Localization of CYS374of Actin by CRYO-EM and Image Analysis of Undecagold-Labeled Filaments
Authors: Milligan R.A. , Whittaker M. , Safer D. 
Published: June 2020

The heavy metal cluster undecagold is a potentially useful reagent for locating surface amino acid residues in three-dimensional maps calculated from electron images. The penultimate residue of rabbit muscle actin - cys374- contains a reactive sulfydryl to which undecagold can be attached via a maleimide-containing linker. Using cryo-EM and image analysis, we have investigated undecagold-labeled F-actin (Au11-actin) and S1-decorated Au11-actin (Au11-actoS1) to demonstrate the general utility of this approach in structural investigations at moderate resolution. In addition, by determining the location of cys374in the actin filament, we sought to provide a valuable constraint for building a high resolution model of F-actin from the x-ray crystallographic structure of the monomer.Rabbit skeletal muscle actin and myosin S1 were prepared by standard procedures. Monomaleimide undecagold was prepared and coupled to actin as described previously. Actin was separated from unincorporated gold by chromatography on Sephacryl S-200. The efficiency of labelling was > 85% as determined by non-denaturing gel electrophoresis.
CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex
Authors: Trinickt John , White Howard 
Published: June 2020

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.
Quantitative Energy-Filtered CRYO-EM of Hydrated Chromosome Fibers, Viruses, and Heavy Atoms
Authors: Langmore John P. , Smith Michael F. , Rankert Debby A. 
Published: June 2020

We have calculated and experimentally measured the extent to which energy filtration improves the S/N in phase- and scattering-contrast images of frozen-hydrated molecules. Multiple scattering calculations based on PW (partial wave) complex scattering amplitudes agree quantitatively with 80 KV image intensities of 50-500nm-thick ice. For an objective aperture of 2.5 nm-1 the PW elastic mean free path is 280 nm. From the ratio of filtered to unfiltered image intensities from 50-500 nm of ice at 80 KV, we calculate an empirical inelastic mean free path of 180 nm. The PW calculations show that Beer's Law should be obeyed for thicknesses up to five mean free paths, whether or not energy filtration is used. Thus the bright-field signal can be used to determine the absolute mass distribution of molecules embedded in thick ice layers.Energy filtration removes background intensity due to inelastic scattering (Fig.1a), and increases the scattering signal from molecules (becoming the sum of both elastic electrons excluded by the objective aperture and inelastic electrons excluded by the energy filter). As the result, the calculated contrast is 2-15 times greater in the Zeiss EM902 than in a TEM without energy filtration (Fig.1b). 80 KeV energy-filtered images of frozen-hydrated molecules should have a higher S/N than unfiltered images taken at intermediate or high voltage (Fig. 1c,d). TMV contrast and S/N is improved by filtration and agrees with calculations (Fig.1b,2). The advantages of energy-filtration are important to the microscopy of chromatin fibers, which are excluded from ice layers ≤200 nm and viruses, which are usually excluded from layers ≤100 nm. Radial density reconstructions from near-focus energy-filtered images of frozen-hydrated chromatin from Thyone sperm show that the centers are not hollow, but very dense, probably containing DNA (Fig.3). Energy-filtered images of icosahedral viruses in even the thinnest possible ice have a much improved S/N ratio, decreasing the dose required for analysis (Fig.2a,b).
Cryoelectron Microscopy of Frozen-Hydrated Pyruvate Dehydrogenase Complexes
Authors: Grassucci Robert , Wagenknecht Terence , Radke Gary A. , Roche Thomas E. 
Published: June 2020

Pyruvate dehydrogenase complexes (PDC’s) from both prokaryotes and eukaryotes exist as large multisubunit assemblies in which one of the component enzymes, dihydrolipoyl transacetylase (E2), forms a structural core that possesses multiple binding sites on its surface for the pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components. We are investigating the structure of PDC’s fromE. coliand bovine heart by cryoelectron microscopy of frozen-hydrated native complexes and various types of subcomplexes prepared in vitro. Fields of frozen-hydrated PDC fromE. coliand bovine heart are shown in Figures 1 and 2 respectively. In many images of theE. coliPDC (M.W. ∽ 5 × 106) the E2 core is visible as a dense square or rectangular mass at the center of the images (see inset in Fig. 1). The E1 and E3 components surround the E2 core, and do not appear to form a symmetrical pattern. Furthermore, there are often apparent gaps of up to several nanometers separating the bound subunits from the surface of the core. These observations suggest that the E1 and/or E3 are linked to the E2 core by flexible polypeptide ‘tethers’ which could be important in the mechanism of active site coupling. Similar observations apply to the images of the larger PDC from bovine heart (M.W. ∽ 9 × 106).Selected images of frozen-hydrated E2 cores isolated fromE. coliand heart PDC are shown in Figures 3 and 4. TheE. coliE2 core consists of 24 identical polypeptides arranged with octahedral symmetry, whereas the larger heart core complex consists of about 60 identical polypeptides apparently arranged with icosahedral symmetry (another component, present in a few copies and referred to as component X, also appears to be integrated into the core complex). Each E2 polypeptide possesses specialized domains (three inE. coli. two in heart) which contain the lipoic acid cofactor and are thought to be involved in coordinating the sequence of reactions catalyzed. TheE. coliE2 cores often appear surrounded by a faint “fuzz” extending out from the surface by about 10 nm which we attribute to the lipoyl-bearing domains. Independent biochemical and biophysical observations are consistent with this interpretation. The larger heart E2 cores exhibit three main image types that probably correspond to views approximately along 5-fold, 3-fold and 2-fold symmetry axes. The fuzzy material observed surrounding theE. colicores is not readily visible in the heart E2, probably because there are fewer lipoyl-bearing domains and because they occupy a much larger volume than inE. coli. Methodologies for characterizing the spatial distribution of the lipoyl-bearing domains by EM, such as labeling them with monoclonal antibodies, are being pursued.
Analysis of Interhexameric Contact Regions in Two Dodecameric Hemocyanins at Subdohain Resolution by Combination of Data from Electron Microscopy, X-Ray Diffraction and Amino-Acid Sequence Studies
Authors: de Haas Felix , van Breemen Jan F. L. , Bijlholt Martha M. C. , van Bruggen Ernst F. J. 
Published: June 2020

Hemocyanin is a biological macromolecule which occurs freely dissolved in the hemolymph of certain invertebrates. The function of this copper containing protein is the transport of oxygen through the organism. In fulfilling this task hemocyanin has developed a similar mechanism as hemoglobin in binding oxygen reversibly and cooperatively. The hemocyanin of arthropods consists of one or more hexamers of subunits with a molecular weight of approx. 75 000. Depending on the species 3-15 types of monomeric subunits occur, which differ in amino-acid composition and in their oxygen binding properties. Each type of subunit fulfills a specific role in the architecture of that hemocyanin. In nature arthropodan hemocyanin is found as a one-hexameric, two-hexameric (dodecameric), four-hexameric or eight-hexameric molecular assembly depending on the species. In this work we focus on the difference in organization of the hexamers in the dodecamer of two different species i.e. the tarantula Eurypelma californicum (a chelicerate) and the crab Cancer pagurus (a crustacean). Eurypelma hemocyanin is made from 7 different subunits called a - g, whereas Cancer hemocyanin consists of 3 subunit types termed α, β, and γ .By image analysis of electron micrographs of the two-hexameric half hemocyanin molecules from Eurypelma and the two-hexameric whole hemocyanin molecules from Cancer, computer averaged projections of these dodecamers were obtained as shown in fig. 1. They differ clearly in their interhexameric contacts. To analyse this difference in more detail these projections were used as a reference in a simulation procedure.