bluuebunny/crossref_metadata_2025 huggingface dataset:
Microstructural analyses of Si3N4 ceramics containing various sintering aids and additional Si3Ni4
Authors: Sung C.M. , Ostreicher K.J. , Buljan S.T. , Baldoni J.G. , Hefter J.
Published: June 2020

Silicon nitride ceramics containing Al2O3 (1.5 wt%)+MgO (4 wt%) (=AM4) and Al2O3 (1.5 wt%) +Y2O3 (6 wt%)(=AY6) sintering aids together with an additional 30 v% Si3N4 have been hot pressed or hot pressed and heat-treated in an attempt to devitrify the glass phase. The expected effect of adding Si3N4 to both these formulations was to modify the glass composition in addition to the overall microstructure of the ceramics while reducing the relative amount of sintering aid. The present investigation was conducted to determine the microstructural and chemical nature of the intergranular phases present.

Investigation of the intergranular phase (IGP) in the AM4-30 v% Si3N4 material indicated that the IGP was generally larger in size than the IGP in AM4 without additional Si3N4.
A TEM study of the formation of the silica structure on gel drying
Authors: Knoester Arie , Afghan Madjid
Published: June 2020

In the manufacture of silica spheres, which are used as a support material for heterogeneous catalysts, partial drying of the silica gel proves to be a crucial step: at this stage the gel undergoes structural changes, which influence the final strength and porosity of the catalyst. Using a special preparation technique, the mechanism of these changes has been studied by means of Transmission Electron Microscopy (TEM).

Silica sphere samples partially dried to different moisture contents as well as a sample of the hydrogel (the undried gel) were prepared with the aim of fixing the pore structures. To this end, the water present in the pores of the samples was replaced by a suitable plastic. This was achieved in consecutive steps: first the water was replaced by ethanol, after which the latter was replaced by a mixture of methacrylate monomers. After polymerization of the monomers ultrathin microtome slices were produced. In some cases the sectioning procedure was hampered by the presence of traces of hygroscopic sodium sulfate. This problem was overcome by introducing a washing step prior to the fixation procedure.
Texture of cholesteric cellulosic gels and films observed by electron microscopy
Authors: Giasson Julie , Revol Jean-François , Gray Derek G.
Published: June 2020

It is now well known that cellulose derivatives form cholesteric liquid crystalline phases in the bulk and in solution. The supramolecular arrangement of the cholesteric materials gives them unique optical properties. Light entering the cholesteric domain is partially reflected. Circularly-polarized light with the same handedness as the helicoidal structure and a wavelength corresponding to the cholesteric pitch will be reflected by the mesophase. Free-standing films displaying a cholesteric order are thus strongly desirable for many different applications.

Even though transmission electron microscopy (TEM) seems to be the perfect technique to extend results obtained by optical microscopy, this approach has been used infrequently in the study of liquid crystals. Liquid crystals don't lend themselves to electron microscopy because of their fluidity. Lyotropic systems are destroyed by evaporation of the solvent under high vacuum. However, under specific conditions, solid cellulosic films can retain a helicoidal organization. Classical embedding and ultra-sectioning can thus be applied on such materials as long as they do not dissolve in water.
Electron microscope studies of the plastic deformation of ternary alloys based on GaAs
Authors: Haswell R. , Bangert U. , Charsley P.
Published: June 2020

A knowledge of the behaviour of dislocations in semiconducting materials is essential to the understanding of devices which use them . This work is concerned with dislocations in alloys related to the semiconductor GaAs . Previous work on GaAs has shown that microtwinning occurs on one of the <110> rosette arms after indentation in preference to the other . We have shown that the effect of replacing some of the Ga atoms by Al results in microtwinning in both of the rosette arms.

In the work to be reported dislocations in specimens of different compositions of Gax Al(1-x) As and Gax In(1-x) As have been studied by using micro indentation on a (001) face at room temperature . A range of electron microscope techniques have been used to investigate the type of dislocations and stacking faults/microtwins in the rosette arms , which are parallel to the [110] and [10] , as a function of composition for both alloys . Under certain conditions microtwinning occurs in both directions . This will be discussed in terms of the dislocation mobility.
Direct Observation of Monolayer Zones in Al-wt 4% Cu
Authors: Jouffrey B. , Dorignac D. , Bourret A.
Published: June 2020

Since the early works on GP zones and the model independently proposed by Preston and Guinier on the first steps of precipitation in supersaturated solid solution of aluminium containing a few percent of copper, many works have been performed to understand the structure of different stages in the sequence of precipitation.

The scheme which is generally admitted can be drawn from a work by Phillips.

In their original model Guinier and Preston analysed a GP zone as composed of a single (100) copperrich plane surrounded by aluminum atomic planes with a slightly shorter distance from the original plane than in the solid solution.

From X-ray measurements it has also been shown that GP1 zones were not only copper monolayer zones. They could be up to a few atomic planes thick. Different models were proposed by Guinier, Gerold, Toman. Using synchrotron radiation, proposals have been recently made.
Characterization of Ultrathin Multilayers for Applications in X-Ray Optics
Authors: Smith David J. , Li Z.G. , Tsen S.-C.Y. , Boher P. , Houdy Ph.
Published: June 2020

Ultrathin multilayers have unique physical properties which are of great practical importance. Our interest here is in layer systems suitable for X-ray optical devices, in particular for X-ray mirrors. Optimization of device performance depends upon such factors as the layer uniformity (thickness and composition), interface roughness, and structural irregularities. Techniques such as Rutherford backscattering, X-ray diffraction and Auger profile analysis provide valuable structural information but averaged over comparatively large specimen regions. High-resolution electron microscopy has been used in the present study to gain complementary insight into the local microstructure of various layer systems, based on combinations of Rh,W,C,B4C, grown by diode rf-sputtering. Samples suitable for electron microscopy were prepared in cross-section using standard grinding, dimpling and argon ion-milling techniques. Most observations were made with a JEM-4000EX HREM operated at 400kV, with selected area electron diffraction (SAED) to determine the average bilayer thickness by reference to the diffraction pattern of the Si (110) substrate. Optical diffractograms (ODMs) and digital processing were used for accurate measurement of the lattice spacings visible in the multilayers.
Dynamical Electron Diffraction in a Crystal with Atoms in Thermal Motion
Authors: Fanidis Christos , Van Dyck Dirk , Van Landuyt Jozef
Published: June 2020

Thermal atom motion destroys the periodicity of the crystal structure. As a consequence electron diffraction patterns (d.p.) and high resolution images will differ from the ones expected from the ideal structure. The changes can originate either from elastic scattering with no energy transferred from or to the agitated crystal or from inelastic scattering with corresponding changes in the state of the scatterer. In the simulations of the high energy electron diffraction the effects are taken into account generally by using the Debye Waller factors (DWf). This treatment is not justifiable because the DWf is deduced from kinematical scattering analysis whereas high energy diffraction is a dynamical process. In the following we attempt to deal systematically with the changes induced in dynamical elastic electron scattering by the thermal atom motion. In order to reduce the calculation time we try to perform the time averaging in the dynamical equations.
Atomic Imaging of Crystals using Large-Angle Electron Scattering in STEM
Authors: Jesson D.E. , Pennycook S. J.
Published: June 2020

It is well known that conventional atomic resolution electron microscopy is a coherent imaging process best interpreted in reciprocal space using contrast transfer function theory. This is because the equivalent real space interpretation involving a convolution between the exit face wave function and the instrumental response is difficult to visualize. Furthermore, the crystal wave function is not simply related to the projected crystal potential, except under a very restrictive set of experimental conditions, making image simulation an essential part of image interpretation. In this paper we present a different conceptual approach to the atomic imaging of crystals based on incoherent imaging theory. Using a real-space analysis of electron scattering to a high-angle annular detector, it is shown how the STEM imaging process can be partitioned into components parallel and perpendicular to the relevant low index zone-axis.

It has become customary to describe STEM imaging using the analytical treatment developed by Cowley. However, the convenient assumption of a phase object (which neglects the curvature of the Ewald sphere) fails rapidly for large scattering angles, even in very thin crystals. Thus, to avoid unpredictive numerical solutions, it would seem more appropriate to apply pseudo-kinematic theory to the treatment of the weak high angle signal. Diffraction to medium order zero-layer reflections is most important compared with thermal diffuse scattering in very thin crystals (<5nm). The electron wave function ψ(R,z) at a depth z and transverse coordinate R due to a phase aberrated surface probe function P(R-RO) located at RO is then well described by the channeling approximation;
High-Resolution Cryo-Electron Microscopy of Two-Dimensional Protein Crystals
Authors: Zemlin F. , Beckmann E.
Published: June 2020

The task in investigating the structure of crystals is to measure the Fourier coefficients in amplitude and phase. Electron crystallography, i.e. electron diffraction combined with electron-microscopical imaging, is a straigthforward method, because there is a priori no “phase problem”. But radiation damage during electron exposure was for a long time an absolutely unbridgeable chasm on the way to high-resolution structure research of two-dimensional protein crystals by electron microscopy. Now, using cryo-electron microscopy and computer image processing, it has been proven possible to overcome this difficulty and achieve high resolution.

Here we present a rough description of the recording procedure. We used a helium-cooled superconducting objective lens with 4.5 K specimen temperature, but the procedure should also be applicable at higher specimen temperatures. The radiation damage is reduced at low specimen temperature, but unfortunately there still remains a remarkable deterioration of the crystal with increasing dose, which means that minimum exposure is advisable.
Structural Study of H,K-ATPase by Electron Microscopy and Image Reconstruction
Authors: Mohraz Manijeh , Sathe Sonal , Smith P.R.
Published: June 2020

H,K-ATPase is an integral protein of the plasma membrane of parietal cells. It is believed to constitute the pump responsible for secretion of acid into stomach. Its catalytic subunit (Mr 110,000) shows striking sequence homology to those of other transport ATPases. Recent studies suggest that there is also a glycoprotein (ca 300 amino acids) associated with the H,K pump, which is very homologous to the β subunit of the Na,K-ATPase.

The enzyme is isolated in protein-rich membrane vesicles from hog stomachs. Formation of two-dimensional crystals is induced in suspensions of the enzyme by methods that had proved successful for crystallization of the Na,K-ATPase.
Unbending: Characterising Distortion and Correcting for IT
Authors: Saxton W O , Duerr R
Published: June 2020

Unbending is becoming the fashionable alternative to simple correlation averaging, just as c-av earlier supplanted Fourier filtration or profile fitting. We note the circumstances in which it may be useful; and we recommend local real space image averaging in preference to Fourier methods.

The displacement field in an image can be estimated reasonably closely from the unit cell positions indicated by cross-correlation with a ‘reference’ image patch. Correlation averaging and unbending both compensate for the displacements of unit cells as a whole, but in their standard forms do not address distortion of individual cells at all. Obviously, both can be easily adapted to do so; some such work has been reported and more is in hand. Experience suggests that correction of the displacements is indeed the most important step: these accumulate from cell to cell across the crystal.
State and Future of Electronic Image Read-OUT in TEM
Authors: Herrmann K.-H.
Published: June 2020

The electron microscope is becoming a link in a highly sophisticated data processing system. The acquisition of image data supplied as a spatial distribution of current density requires a position sensitive electron detector which converts the current into digital information to be processed by image storages and computers to retrieve the information in which the user is interested. The ultimate goal of this interface is a lossless conversion with respect to both the number of pixels and the detection quantum efficiency (DQE) as well as high speed, minimum distortion, and linearity. I shall try to outline the present state of image read-out using both the conventional photoplate with spatial digitizing equipment and conventional TV sets. Subsequently it will be discussed how the future CCD technology (charge coupled device) may overcome some restrictions of present solutions.

Every spatial recording device combines a conversion with a storage function in order to build up a signal-to-noise ratio (SNR) sufficient for detection. The following characteristics describe their performance:
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
Point Resolution Below 1 Å by Means of Electron Holography?
Authors: Völkl Edgar , Lichte Hannes
Published: June 2020

The optical resolution limit δD according to Lord Rayleigh is given by δD = 0.61 λ/α0 where an is the aperture of the objective lens and λ the wavelength. Assuming identical conditions in an electron microscope, one would obtain (depending on the accelerating voltage) a resolution of several 0.001 nm. However, the actual resolution limit is found to be worse by a factor of a hundred. This is due to the aberrations of the objective lens. Generally, the image wave if>(x,y) is given by the convolution of the object wave ψx,y) with the Fourier transform of the wave transfer function exp(ix) (assuming isoplanacy),
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.
Electron Microscopy of Negatively Stained and Frozen-Hydrated Bacteriophage Φ29
Authors: Olson N. H. , Xu W. , Grochulski W. D. , Andersont D. L. , Baker T. S.
Published: June 2020

Bacteriophage Φ29 is a complex-shaped, double-stranded DNA virus that infects Bacillus subtilis . Mature Φ29 particles contain six major structural proteins which give rise to several distinctive features: a prolate-shaped head (gp8), fibers (gp8.5) which radiate from the head, a connector-collar region (gp10 and gp11), collar appendages (gp12*) and a tail (gp9). Although much is known about Φ29 biochemistry, genetics and assembly, relatively little is known about its detailed structure. Three-dimensional reconstructions of the collar demonstrated that it contains two structurally distinct features: an upper portion (proximal to the head) with 12-fold axial symmetry (gp10) and a lower protion with 6-fold symmetry (possibly both gp10 and gp11). The capsid is believed to be organized with two apical regions with trimeric aggregates of gp8 dimers arranged on a T=l icosahedral lattice separated by 10 additional trimers in the equatorial region. Φ29 proheads are assembly intermediates that contain only gp8, gp8.5, gp10 and the scaffolding protein (gp7) which is released upon DNA encapsidation. We have imaged Φ29 with conventional negative-staining and the recently developed cryo-microscopy techniques with the goal of examining the structural basis for Φ29 morphogenesis.

Vitrified Φ29 proheads display distinct outlines with one end pointed and the other somewhat flattened and they often orient with their long axes in the plane of the vitreous water (Fig. 1). Occasional end-on views with a circular profile are also observed. Proheads show a tendency to associate in pairs with the closest contact between the flattened ends (Fig. 1, arrow-head). Longer morphological variants are also occassionally observed (Fig. 1, arrow). The morphology of proheads is dramatically distorted in negative-stain preparations indicating significantly poorer preservation using this technique (Fig. 2). Proheads are also permeable to stain.
The Structure and Subunit Organization of the Bacterial Flagellar Motor by Scanning Transmission Electron Microscopy and Cryoelectron Microscopy
Authors: Sosinsky Gina E. , Francis Noreen R. , DeRosier Charles D. , DeRosier David J. , Hainfeld James , Wall Joseph
Published: June 2020

The bacterial flagellum is unique in having a rotary motor. In Salmonella typhimurium, the basal body, a component of the motor, consists of four rings (denoted M, S, L, and P) threaded on a coaxial rod. The M, L, and P rings are each composed of a different protein: FliF=61 kD, FlgH=22 kD, and FlgI=36 kD, respectively. The rod contains at least four different proteins: FlgB=15 kD, FlgC=14 kD, FlgF=26 kD, and FlgG=28 kD. Using quantitative gel analysis, Jones et al. estimated that there are about 26 copies of FlgG, FlgH, Flgl and FliF, and 6 copies of FlgB, FlgC and FlgF per basal body. The total mass of these 7 proteins per basal body is ∽4200 kD. There appear to be additional proteins in the basal body, but their locations and amounts are not known. Our aim is to produce subcomplexes of the basal body and determine their structures and masses using electron microscopy. This approach is complementary to that of Jones et al. and can reveal the presence and amounts of as yet unidentified components. We find, in pH3- or pH4-treated preparations of basal bodies, four subcomplexes of the hook basal body complex (HBB): the HLPRS (hook, L and P rings on the distal rod, proximal rod, S ring); the HLPR (lacks the M and S rings), the HLP (lacks the M, S, and proximal rod); and the LP complex (Figs. 1 and 2). We have been able to visualize the three-dimensional structure and the subunit organization using the combined techniques of cryoelectron microscopy and image analysis. These studies suggest that the S ring is a separate component from the rod or M ring and that the rod consists of two sections. Because the different sub-complexes are distinguishable in a field of particles, we measured the molecular masses of the individual subcomplexes using the Brookhaven STEM even though these preparations are not homogeneous (Fig. 3). All the structures analyzed so far had hooks attached. We measured the length and mass/length from STEM images and then subtracted the mass of the hook. Preliminary results show that the molecular mass of the hookless basal body is 4400−500 kD (n=165), that of the LP-rod (proximal and distal) is 3500±300 kD (n=52), and that of the LP-distal rod is 2300±450 kD (n=76) (Fig. 4). The difference between these three molecular weights gives estimates of the mass of the M and S rings (4400 - 3500 = 900 kD) and proximal rod, 3500 − 2300 = 1200 kD. The mass of the M and S rings may be underestimated due to the undetected presence of HLPRS subcomplexes in the basal body data set. We are presently measuring and re-evaluating masses for the subcomplexes in order to get more accurate estimates of the masses and numbers of subunits.
Three-Dimensional Image Reconstruction of the 50S Subunit from

Escherichia Coli

Ribosomes Lacking 5S-rRNA
Authors: Radermacher Michael , Nowotny Volker , Grassucci Robert , Frank Joachim
Published: June 2020

In earlier studies the structure of the 50S ribosomal subunit from E. coli has been determined from electron micrographs, using the single exposure random conical reconstruction technique. For the understanding of the function of ribosomes the single proteins and ribosomal RNAs need to be located within the ribosome structure. For localization of most proteins immunoelectron microscopy and neutron scattering data are available. The current study’s goal is the localization of the 5S rRNA from a comparison of the structures of complete 50S subunits with that of subunits reconstituted omitting the 5S rRNA.

By Phenol extraction of purified 50S subunits the rRNA fraction was separated form the total protein fraction (TP50). This rRNA fraction was separated into 23S rRNA and 5S rRNA via HPLC on a DEAE-column. The total reconstitution of 23S rRNA and the equivalent amount of TP50 resulted in particles lacking the 5S rRNA. For electron microscopy the subunits were prepared in a negative stain sandwich preparation.

Twenty tilt pairs at 50° tilt and 0° were recorded, with an electron dose of approximately 10el/A2 and a magnification of 49,000. A total of 983 particles were selected from these data. The 0° images were aligned using the procedure described in.
A Simple Scanning Tunneling Microscope with Very Wide Scanning Range
Authors: Yao J.E. , Shang G.Y.
Published: June 2020

Scanning Tunneling Microscope (STM) has been a powerful tool for study of surfaces in the range of about 1 micrometer. The small field of view is enough for imaging homogeneous surfaces with atomic or near-atomic resolution. If, however, integrated circuits, gratings and other small “man-made” structures have to be observed, a STM with very wide scan range, for example, 10 to 100 micrometers is needed. In most of the STMs currently in use, three-dimensional scanner are fabricated from piezoceramic stacks, tubes and beams. The maximum scanning range is restricted to about a micrometer because of the maximum allowable control voltage and piezo element dimensions. Recently, Takashima Koshi has constructed a x/y scan stage for observation of grating(1). In a similar point of view, We have designed and built a simple scanner (Fig.1), which includes a base B, a mechanical amplifying device (consisting of a spring lever S and a metal tube M), x/y driving elements D, z control piezo tube P and tip T. The relation between the displacement dx(dy) and applied voltage V for the scanner is described by the equation:

dx(dy)=KV(2L+l)/2d. Where, K is the voltage sensitivity in nm/v; L and l are the lengths of M and S respectively; d is the distance between the axis of S and that of D. When L=30mm, l =8mm, d=5mm, k=60nm/v, a scan range of 120μm will be obtained.
Scanning Tunneling Microscopy of Amorphous Carbon Films
Authors: Fotino Mircea , Parks D.C.
Published: June 2020

In the last few years scanning tunneling microscopy (STM) has made it possible and easily accessible to visualize surfaces of conducting specimens at the atomic scale. Such performance allows the detailed characterization of surface morphology in an increasing spectrum of applications in a wide variety of fields. Because the basic imaging process in STM differs fundamentally from its equivalent in other well-established microscopies, good understanding of the imaging mechanism in STM enables one to grasp the correct information content in STM images. It thus appears appropriate to explore by STM the structure of amorphous carbon films because they are used in many applications, in particular in the investigation of delicate biological specimens that may be altered through the preparation procedures.

All STM images in the present study were obtained with the commercial instrument Nanoscope II (Digital Instruments, Inc., Santa Barbara, California). Since the importance of the scanning tip for image optimization and artifact reduction cannot be sufficiently emphasized, as stressed by early analyses of STM image formation, great attention has been directed toward adopting the most satisfactory tip geometry. The tips used here consisted either of mechanically sheared Pt/Ir wire (90:10, 0.010" diameter) or of etched W wire (0.030" diameter). The latter were eventually preferred after a two-step procedure for etching in NaOH was found to produce routinely tips with one or more short whiskers that are essentially rigid, uniform and sharp (Fig. 1) . Under these circumstances, atomic-resolution images of cleaved highly-ordered pyro-lytic graphite (HOPG) were reproducibly and readily attained as a standard criterion for easily recognizable and satisfactory performance (Fig. 2).