bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of a 400KV Ultrahigh-Resolution Analytical TEM
Authors: Tsuno H. , Honda T. , Kokubo Y.
Published: June 2020

The condenser-objective (C/O) lens proposed by Riecke, which has a very short gap length and small spherical aberration, was utilized for a commercial 200 kV ultrahigh resolution analytical TEM by Yanaka and Kaneyama. Fig. 1 shows the relation between theoretical resolution and objective lens (OL) spherical aberration coefficient (Cs) at accelerating voltages 200-1250 kV. It was reported that the Cs of a 400kV high resolution TEM is 1.0 mm and its resolution is 0.167 nm. The Cs of 400kV analytical TEM is 1.8 mm and the pre-field spherical aberration coefficient (Csp) is 1.8 mm. Fig. 2 (A), (B) show beam broading in specimens against the thickness when a 200kV and a 400kV electron beam transmit the specimen (C-Au), respectively. The broading of 400kV electron beam is about half of 200kV one. Then it is expected that spacial resolution of x-ray analysis improve. The above-captioned 400kV ultrahigh resolution analytical TEM is designed by applying a new technology which is adopted for a 200kV ultrahigh resolution analytical electron microscope, JEM-2010.

Its fundamental construction is the same as the 400kV analytical electron microscope JEM-4000FX, except the 0L. The goniometer is a modified JEM-2010 goniometer, because it is too small for 400kV EM. Although it was expected that the focus ampere turn increases because of its short gap length, the objective lens coil used by JEM-4000EX/FX is adopted, because it has enough capacity. The shapes of the upper yoke and objective polepiece were calculated by the finite element method (55×110 Meshes) under the following condition: (1) maximum tilting-angle 10° (2) x-ray take-off angle 17.5° and solid angle 0.068 strad (3) minimized Cs.
Development of an Autofocus System in Tem
Authors: Ito T. , Kondo Y. , Mori S. , Watabe T. , Arai Y.
Published: June 2020

An autofocus system has been developed by combination of a high-sensitivity TV camera, a 32-bit CPU with a frame memory, and TEM image wobbler function. Center-crossing line profiles of images, instead of 2D images, have been used for autofocusing, thus ensuring high speed autofocusing within 8∽24 seconds for the medium magnification range, × 2000 ∽ 50000.

The image wobbler is a well-known function for a standard commercial TEM, and widely used for biological specimens. For autofocusing by using an image wobbler, the 2D image matching technique is extremely important, and some papers have been published. However, in image matching, image data has to be considered as large as 512×512×8 bits, thus requiring a long computation time. A new type of matching, line profile matching instead of image matching, can save much time and computer memory.
A New Computer Control System for the EM 910 Transmission Electron Microscope
Authors: Benner Gerd , Prinz Manfred , Bihr Johannes , Frey Josef
Published: June 2020

Modern analytical electron microscopes must provide a multitude of illumination and imaging modes, a user-friendly operation and a high degree of flexibility. In addition a large number of monitoring and control functions must be performed. In the new Zeiss EM 910 this is achieved by a complete digitization of the instrument control system. The computer network comprises an AT-compatible system computer, 4 microprocessor-controlled subsystems and a flexible data and program memory. Two control panels and an interactive control monitor are used for operation of the instrument. A keyboard is integrated for data input.

Fig. 1 shows a block diagram of the computer control of the EM 910. The integrated system computer with an 80286 processor and a clock frequency of 12 MHz controls and monitors the electron optics (lenses, deflection systems, stigmators). Specially developed interrupt-controlled parallel interfaces ensure rapid communication between the system computer and the 4 autonomous Z80 microprocessor-controlled subsystems. The subsystems are:

1. gun subsystem which controls and monitors the high-voltage system;

2. goniometer subsystem for control and operation of the motorized 4-axis eucentric goniometer;

3. camera subsystem which controls the sheet film camera and the components required for exposure such as automatic screens and the shutter;

4. vacuum subsystem for control of the vacuum system and monitoring the compressed air and water supply.
<310> Single-Crystal LaB6 as Thermal Field Emitter for Electron Microscope
Authors: Harada Ken , Nagata Haruto , Shimizu Ryuichi , Tanji Takayoshi , Yada Keiji
Published: June 2020

Thermal field emission (T.F.E.) properties of single crystal LaB6 -tips has been investigated by observing emission patterns. Applying field evaporation technique we succeeded to get the clean pattern consisting of <310> spots with very good reproducibility. This investigation has led to conclusion;, the <310> spot is promising electron source of high brightness provided that the tip is operated at tip temperature∽ 1000-1050°C in vacuum of 10−9 Torr region.

As a preliminary experiment of brightness-measurement, we mounted the <310> LaB6-tip in a commercial type TEM, JEM-100CX-FEG, attached with an electron gun system for T.F.E. of <100> W-tip, being operated at 10−9 Torr region without Schottky shield electrode. The LaB6-tip, however, can not be operated without the Schottky shield because thermionic emission (T.E.) from the LaB6-tip is considerably high even though the tip is operated at lower than ∽1000 °C. In the present experiment, therefore, we manufactured a Schottky shield electrode as shown in Fig.l and performed the measurement of brightness by setting the Schottky shield electrode, applied the same voltage as the tip since the electron gun system has no extra feed-throughs for bias-voltage.
Novel Methods of Microanalysis Employing Secondary- and Auger-Electron Spectroscopy in Stem
Authors: Kruit P.
Published: June 2020

Introduction: Electron spectroscopy is a well established technique in physics and material science and merely adding the spatial resolution of a STEM would already open a new field of applications. However, by using the specific advantages of electron microscope optics and the expertise in thin specimen preparation of microscopists, electron spectroscopy in the STEM can yield information that cannot be obtained in any other way.

Instrumentation: The feasibility of extracting a large portion of the Auger and secondary electrons from the magnetic field of an immersion condenser objective lens has now been demonstrated. The scheme involves a careful shaping of the magnetic field through which the electrons are guided to a deflector which separates the secondary beam from the primary beam. The parallelizing action of this field forces the electrons into a beam of small opening angle which can be accepted by traditional optics including an electrostatic energy analyser. Results reported in this paper are from a prototype instrument; energy analysers mounted on fully ultra high vacuum microscopes are now also in operation or will become operational very soon. To detect the secondary or Auger electron together with the primary electron which was responsible for its emission requires only small additions to the instrumentation. Single electron sensitivity in both the secondary electron detector and the EELS detector are of course necessary, but this can be done with standard detectors with a timing resolution of better than 5 ns. Commercial electronic equipment can then sort the coincident events.
Stochastic Limitations to Phase and Contrast Determinations in Electron Holography
Authors: Lenz Friedrich , Völkl Edgar
Published: June 2020

An important step in the evaluation of electron holograms consists in the determination of contrast μ> 0 and phase φ of a distribution function

recorded using a large but finite number N of electrons. The direction of the interference fringes and their period (‘fringe width’) which is used as length unit are assumed to be known. The x axis runs perpendicular, and the y axis parallel to the fringes. It is assumed that a rectangular area is evaluated, the width of which in x direction is an integer multiple of the fringe width, and that within this area the fringe system is parallel and equidistant, i.e. μ and φ are constant. We shall restrict ourselves to the interval 0 < x ≤ 1. According to (1), the probability for an electron to be recorded within an interval between x and x + dx is W(x, φ,μ) dx. During the practical evaluation of a holographic fringe pattern, however, the x values are not measured but only the numbers of electors recorded within finite sampling areas.
Subunit Arrangement of and Rearrangements in E. coli F1F0 ATP Synthase Determined by Cryoelectron Microscopy
Authors: Gogol Edward. P. , Capaldi Roderick. A.
Published: June 2020

The F1F0 ATP synthase is the enzymatic complex which interconverts the electrochemical energy of the transmembrane proton gradient, generated by oxidative phosphorylation of metabolic substrates, into the energy currency of ATP. In doing so, it reversibly couples transmembrane proton flow to the catalysis of the high energy terminal phosphate bond of ATP. Even the simplest example of the enzyme, the bacterial F1F0, is a complex assembly of eight subunits in non-unit stoichiometry, five making up the catalytic F1 portion (α3β3γδε) and three the F0, the proton channel (a b2c10‒12) We are attempting to understand the role of subunit interactions in the coupling phenomenon and the catalytic mechanism, which exhibits an extraordinarily high degree of allosteric cooperativity.

Three-dimensional reconstruction of negatively stained arrays of the F1 has shown it to be a hexagonal barrel-shaped structure; cryoelectron microscopy of unstained, frozen-hydrated specimens (figure 1a) has revealed an interior cavity partially occluded by an internal structure.Immunolabelling with monoclonal Fab fragments to the α subunit, followed by alignment and averaging of cryoelectron images, has demonstrated the alternating αβ arrangement of subunits around the periphery of the F1. The internal structure has also been localized next to one of the (catalytic) β subunits (see figure 2a,b). Addition of Fab fragments to the carboxy-terminal part of the (single-copy) γ subunit preserves the characteristic triangular appearance and uniform orientation of the Fl-Fab complexes (figure lb).Averages of these images place the additional density of the label at the periphery of the structure, superimposed on a β subunit (figure 2c). Surprisingly, similar results are obtained with readily-binding monoclonal antibodies to each of the single-copy subunits.
T4 Replication Accessory Proteins Visualized as Complexes with DNA by Cryoelectron Microscopy
Authors: Gogol Edward P. , Young Mark C. , Kubasek William L. , Jarvis Thale C. , von Hippel Peter H.
Published: June 2020

Replication of bacteriophage T4 is accomplished by a set of proteins encoded by the T4 genome.Central to replication is the DNA polymerase, the product of gene 43 (g43p); while it contains the intrinsic functions of template-directed DNA synthesis, it requires a set of accessory proteins as a minimal system to function with a rate, fidelity and processivity comparable to those obtainedin vivo.These accessory proteins are g44p, g45p and g62p; g32p, a single-stranded DNA binding protein, also assists in the replication process, bothin vitroandin vivo.In the absence of DNA, g44p and g62p form a 4:1 complex which has a DNA-dependent ATPase activity; g45p is a trimer in solution. Their association state when bound to DNA is however unknown, as is the mechanism by which they dramatically improve the replication activity of g43p.

We have examined the complex of the three replication accessory proteins and g32p as a complex with DNA by cryoelectron microscopy, avoiding stains or fixatives which may disrupt the labile structures formed. Under conditions which promote the polymerase-enhancing activity of this four-protein system, distinct "hash mark" structures are visible along strands of replicative form M13 DNA (figure 1). These structures are formed only in the presence of all four proteins and ATP; absence of any one of the proteins or ATP, or substitution of non-hydrolyzable ATP analogues, fails to yield any of the hash marks.
Description of two regular structures associated to the cell envelope of Thermus thermophilus: a structural transformation?
Authors: Carrascosa J.L. , R. Castón José , Faraldo M.M. , Berenguer J. , de Pedro M.A.
Published: June 2020

Thermus thermophilus HB8 is a Gram-negative thermophilic eubacteria, with a well-defined outer membrane and a thin peptidoglycan layer around the cytoplasmic membrane. There is a S-layer on the cell envelope, whose main component is a protein of Mr around 100.000, masked by a lipopolysaccharide-like outer surface that can be revealed by freeze-fracture and shadowing of the native surface of the cells of T. thermophilus.

When membrane fractions of this bacteria are treated with Triton X-100 in presence of EDTA, two-dimensional crystals with a hexagonal ordering could be observed by negative staining (figures 1a, 1b). This structure, called S1, had an unit cell dimensions a = b = 20 nm. Nevertheless, if the treatment is developed without the quelant agent, a structure with p2 symmetry is observed, S2, (figures 1d, 1e) with a = 24 and b = 21 nm. We have characterized both crystals by electron microscopy and two-dimensional analysis by Fourier methods. The resulting images showed very different characteristics; while the p6 structure is a typical S-layer, fig. 1c, the other one is a porin-like or regular outer membrane protein structure, fig. 1f, (2).This is surprising because both fractions contains the same ˝P100˝ protein (revealed by biochemical analysis).
Energy-Filtered Electron Microscopy (EFEM) of Frozen Hydrated Biological Specimens
Authors: Probst Wolfgang , Zellmann Erhard , Bauer Richard
Published: June 2020

The preparation of hydrated biological specimens for the use in a TEM has made a great stride foreward due to the work of Dubochet et al. on vitrification and Muller et al. on high pressure freezing. Transfer units and cryo stages for the microscopes allow imaging of specimens in the 100K range. Due to simple physical reasons, however, contrast of such kinds of specimen is still a problemm in conventional transmission electron microscopes (CTEM). Solutions as they are provided by an EFEM will be shown and explained in the following.

Ice is the main constituent of frozen hydrated specimens. The large ratio of inelastic-to-elastic total cross section of 4.0 in case of ice which is even more than that for carbon results in an unavoidable high amount of inelastically scattered electrons. Blurred images and lacking contrast are due to that fact. The EEL spectra from a frozen hydrated section of biological material before and after freeze drying in the microscope document this fact. (Figure 1). Increased scattering probability and thickness contribute to the inelastic loss. In Figure 2 the EEL spectrum from a thin pure ice layer without any support is compared to the spectrum from thin freeze dried cryo section on a thin support. In case of ice the maximum of the low loss range is clearly shifted towards zero loss, mainly due to oxygen low loss and plasmon and to hydrogen core loss. Thus for the images shown in the following Figures a narrow energy window of 10 eV is used really to cut off all the inelastically scattered electrons.
Ultrastructure of Two Mutants in the H-Subunit of Recombinant Human Ferritin: Ordered Arrays Indicate Altered Surface Properties
Authors: Massover William H. , Levi Sonia , Luzzago Alessandra , Tateo Paola , Arosio Paolo
Published: June 2020

Two major types of polypeptide subunits, H and L, coassemble (n = 24) as heteropolymers to form molecules of the iron-storage protein, ferritin [1]. The significance of the H-type subunits for molecular structure and function remains unclear. The present study has examined the ultrastructure of recombinant human ferritins which are homopolymers of wild-type or mutant H-subunits [2]. Both mutations change the aminoacid sequence along the outer surface of the molecule; 9Cd has a single substitution of glycine for the lysine at position 86, and Ml has a duplication of 18 aminoacids [2].

Recombinant ferritins were overexpressed in E. coli. and purified as described by Levi et al [2]. Samples (0.1-0.5 mg/ml) were deposited onto a thin carbon film stripped from freshly cleaved mica and carried on 400 mesh grids; these specimen carriers had been made hydrophilic by glow discharge treatment. After negative staining with 2% sodium silicotungstate (pH 7.0), the specimens were dried and examined at lOOkV with a JEOL 100CX electron microscope.
Studies of Ge/Si(100) and Observation of Atomic Steps on Si(100) using Biassed Secondary Electron Imaging in a UHV STEM
Authors: Krishnamurthy Mohan , Drucker Jeff S. , Venablest John A.
Published: June 2020

Secondary Electron Imaging (SEI) has become a useful mode of studying surfaces in SEM[1] and STEM[2,3] instruments. Samples have been biassed (b-SEI) to provide increased sensitivity to topographic and thin film deposits in ultra high vacuum (UHV)-SEM[1,4]; but this has not generally been done in previous STEM studies. The recently developed UHV-STEM ( codenamed MIDAS) at ASU has efficient collection of secondary electrons using a 'parallelizer' and full sample preparation system[5]. Here we report in-situ deposition and annealing studies on the Ge/Si(100) epitaxial system, and the observation of surface steps on vicinal Si(100) using b-SEI under UHV conditions in MIDAS.

Epitaxial crystal growth has previously been studied using SEM and SAM based experiments [4]. The influence of surface defects such as steps on epitaxial growth requires study with high spatial resolution, which we report for the Ge/Si(100) system. Ge grows on Si(100) in the Stranski-Krastonov growth mode wherein it forms pseudomorphic layers for the first 3-4 ML (critical thickness) and beyond which it clusters into islands[6]. In the present experiment, Ge was deposited onto clean Si(100) substrates misoriented 1° and 5° toward <110>. This was done using a mini MBE Knudsen cell at base pressure ~ 5×10-11 mbar and at typical rates of 0.1ML/min (1ML =0.14nm). Depositions just above the critical thickness were done for substrates kept at room temperature, 375°C and 525°C. The R T deposits were annealed at 375°C and 525°C for various times. Detailed studies were done of the initial stages of clustering into very fine (∼1nm) Ge islands and their subsequent coarsening and facetting with longer anneals. From the particle size distributions as a function of time and temperature, useful film growth parameters have been obtained. Fig. 1 shows a b-SE image of Ge island size distribution for a R T deposit and anneal at 525°C. Fig.2(a) shows the distribution for a deposition at 375°C and Fig.2(b) shows at a higher magnification a large facetted island of Ge. Fig.3 shows a distribution of very fine islands from a 525°C deposition. A strong contrast is obtained from these islands which are at most a few ML thick and mottled structure can be seen in the background between the islands, especially in Fig.2(a) and Fig.3.
High-Resolution Plan View Imaging of Clean and Metal Adsorbed Si Surfaces
Authors: Ozawa S. , Kobayashi K. , Tanaka Y. , Koike H. , Yagi K.
Published: June 2020

Surface electron microscopy either in TEM or REM mode has attracted much attention. High resolution (HR) imaging of surfaces in atomic resolution level in profile or plan view imaging mode [1,2] uses surfaces of fine particles grown on peripheries of substrates. Thin self-support films with clean surfaces are generally difficult to make. Therefore, previous observations on clean Si(111)7×7 surfaces [3,4] could resolve only basic lattice fringes of the 7×7 lattice. No details of the unit cell structure have been reported.

Scanning Tunneling Microscopy (STM) could reveal details of the 7×7 structure together with electronic structures [5,6]. However, STM is difficult to use for studies of surface dynamic processes. Therefore, HR-TEM is still an important technique. In the present report, HR-TEM observations of clean and metal adsorbed Si(111)7×7 and (001)2×1 with use of a 1 MV HR-UHV-EM [1,2,7] are described. An advantage to use the 1MV electron microscope is to decrease inelastic scattering as in HR-REM observations of Si(111) surfaces [8].

The specimens of Si (111) and (001) crystal with thin parts were prepared by a method described previously [9]. They were clamped in a side-entry holder used in a previous REM study [8]. Thinning to a thickness transparent to the electrons were carried out by in-situ flash heating at around 1200°C.
Crystallographic Features of PdO Patches Formed from Pd Film
Authors: Ye H.Q. , Ning X.G. , Smith D.J.
Published: June 2020

Recently HREM observations of small particles and extended gold as well as oxide surfaces indicated that profile imaging could provide atomic scale information about the morphology of materials. Later studies for other novel metals, such as Ag and Pd, have been reported to oxidate-some surface oxides at vacuum conditions of the used electron microscope. In this presentation, we describe the crystallographic feature of PdO, which was the production of surface reaction during profile imaging observations.

The clean surface under intereaction of the incident beam with Pd crystal could obviously result in chemical changes. The series images in Fig.1 of a (001) surface of the Pd crystal(region R) and the edges of holes (P and Q) in the [110] Pd foil illustrate oxide formation at the surface. In Fig.1a, a PdO patch was overlapped onto the region R with rather faint contrast, where the unit cells of the newly formed crystalline and the Pd matrix were outlined. In other hand, two holes P and Q were still full with the amorphous contamination and the surface close to these regions did not changed. After a few minutes, the [001] PdO lattice image became sharp and clear, and then dominated most of the region R (Fig.1b). It was seen that a [111] PdO patch with slight expended lattice spacings and exactly parallel orientation relationships was generated in the region P once carbonaceous materials was removed (Fig. 1b and c). In Fig.1c an [010] PdO patch was well developed in the region Q.
Design of a Spectroscopic Low-Energy Emission Microscope
Authors: Veneklasen Lee H.
Published: June 2020

This paper discusses some of the unique aspects of a spectroscopic emission microscope now being tested in Clausthal. The instrument is designed for the direct parallel imaging of both elastic and inelastic electrons from flat surfaces. Elastic contrast modes of the familiar LEEM include large and small angle LEED, mirror microscopy, backscatter diffraction contrast (for imaging of surface structure), and phase contrast (for imaging of step dynamics)(1). Inelastic modes include topology sensitive secondary, and work function sensitive photoemission. Most important, the new instrument will also allow analytical imaging using characteristic Auger or soft X-ray emissions. The basic instrument has been described by Bauer and Telieps (2). This configuration has been redesigned to include an airlock, and a LaB6 gun, triple condensor lens, magnetic objective lens, a double focussing separator field, an imaging energy analyzer, and a real time image processor.

Fig. 1 shows the new configuration. The basic beam voltage supply Vo = 20 KV, upon which separate supplies for the gun Vg, specimen Vs, lens electrode Vf, and analyzer bias Vb float. The incident energy at the sample can be varied from Vs = 0-1 KV for elastic imaging, or from Vg + Vs = (3 + Vs) KV for inelastic imaging. The image energy window Vs±V/2 may be varied without readjusting either the illumation, or imaging/analyzer optics. The diagram shows conjugate diffraction and image planes. The apertures defining incoming Humiliation and outgoing image angles are placed below the separator magnet to allow for their independent optimization. The instrument can illuminate and image 0.5-100 μm fields at 0-1 keV emission energies with an energy window down to 0.2 eV.
Single-Pole Objective Lens for Low-Voltage SEM High Resolution Wafer Observation
Authors: Yonezawa Akira , Takeuchi Yukio , Kano Takeshi , Ishijima Hiroshi
Published: June 2020

The low-voltage in-lens FE-SEM can observe a 10nm diameter pinhole on Si polycrystal film and etch residue which cannot be observed by a typical SEM. However,this in-lens SEM cannot observe a large size specimen such as a wafer. A new single pole objective lens was recently designed for this purpose. Because of the small bore diameter (2∼3mm),when using this lens,a negative potential must be given to the specimen holder to improve the efficiency of the secondary electron detection.

From researching of a single pole magnetic lens system used for observing a wafer with high resolution, we obtained the secondary electron image. Fig.1 shows the single pole objective lens mounted on the specimen chamber. L is the distance between the pole face and the opposite iron wall,R is the radius of the outer yoke,and WD is the working distance. The design features are as follows:
Low-Voltage Scanning Electron Microscope (LVSEM) with a Single-Polepiece Lens
Authors: Müllerová I. , Lenc M.
Published: June 2020

The advantages of the LV SEM are well known. Recently a lot of interesting results from this field were presented which were obtained thanks to development of field emission guns and to the enourmous progress in the computation techniques in electron optics.

One of the simplest arrangements of the LVSEM is shown in Figure 1. The Tesla SEM BS 350 with a field emission gun and the TF-W/100-Zr cathode was used for our experiment. The gun provides 10−10 A current in the diffraction limited spot (for the angular density 0.20mA sr−1). If a potential Usp is applied to the specimen the energy E of the electrons that strike the specimen is Ep-eUsp (Ep-primary beam energy, e-elementary charge). The produced secondary (SE) and backscattered (BSE) electrons are accelerated towards the semiconductor detector by the electrostatic field and their energy spectrum extends from eUsp to Ep. The final energy of the SE and BSE can then be sufficient for achieving a reasonable amplification of the semiconductor detector which is directly proportional to the energy of the electrons that strike the detector. We calculated optical properties for a combination of the electrostatic and magnetic lenses of the basic geometry shown in Figure 1 and for an arrangement with the single polepiece lens shown in Figure 2. We particularly investigated coefficients of the chromatic (Cc) and spherical (Cs) aberrations as functions of the ratio of the primary beam energy to the energy of the electrons that strike the specimen Ep/E for some optimum position of the specimen, electropstatic and magnetic field. Our results are shown in Table 1. The coefficients Cs and Cc do not change with the energy Epor E if the ratio Ep/E is maitained the same and aberrations are lower for larger ratios Ep/E, so that the influence of the contribution of the electrostatic lens aberrations is negligible for our geometry. For example, if we require a resolution limit r=2nm and an energy of the electrons that strike the specimen E=300eV, it is possible to calculate that the coefficient of the aberrations must be Cs<0.21mm and Cc<0.14mm for an energy width AE=0.2eV, so that we need the ratio Ep/E≥150 for the arrangement shown in Figure 1 (i.e.Ep≥45keV) and Ep/E≥33 for the arrangement shown in Figure 2 (i.e.Ep≥10keV).The advantages of the combination of the magnetic lens with the electrostatic cathode lens for the high resolution very low energy electron microscopy are well known . We assume that for the LVSEM only a medium electrostatic field strength is admitted at the specimen surface. Nevertheless, our experimental arrangements should certainly be optimized in the future.
Fast Fourier Transform Techniques for Measuring SEM Resolution
Authors: Dodson A. , Joy D. C.
Published: June 2020

Precise knowledge of a particular SEM's resolution is important for at least two reasons to the microscopist operating the instrument. First, accurate interpretation of micrographs obtained in the course of daily operation depends on knowing the performance limits of the instrument. Second, it is important for the microscopist to be able to verify instrumental performance.

Spatial resolution is typically determined using micrographs of materials containing structures having a “known” spacing. By showing that two objects in the micrograph are separated by some distance, it is claimed that the instrument is capable of resolving this spacing. This method is passable for instruments whose maximum resolution resolves only features of known spacing. However, for High Resolution SEM's (HRSEM) this method is not adequate. It is difficult to identify specimens with surface structure that can be verified to exist at the ultimate limits of today's HRSEM resolution. The fact that verifying resolution by this method depends on being able to verify the spacing of surface structure on a particular specimen leads to an interesting paradox - the resolution of a given instrument can be verified only after a better instrument is available to characterize the structure spacing.
Secondary Electron Image Contrast in the Scanning Electron Microscope
Authors: Suga Hiroshi , Fujiwara Takafumi , Kanai Nobuhiro , Kotera Masatoshi
Published: June 2020

An image contrast given in the scanning electron microscope(SEM) is due to differences in a detected number of secondary electrons (SE) coming from the specimen surface. The difference arises from the topographic, compositional and voltage features at the specimen surface. Two kinds of approaches have been taken for the quantification of SE images. One is to simulate electron trajectories in vacuum toward the detector, assuming the typical angular and energy distributions of electrons emitted from the specimen surface. However, the typical angular and energy distributions are not always applicable if a topographic or a compositional feature is present at the surface. The other is to simulate electron trajectory in the specimen. It is possible to obtain angular, energy, and spatial distributions of electrons emitted from the specimen surface. However, in order to discuss the SEM contrast based on these data, one has to assume that, for example, all slow electrons (<50eV) may be collected by the SE detector, or fast electrons ((>50eV) electrons may take a straight trajectory in the vacuum specimen chamber of the SEM. In a practical SEM picture of, for example, an etch-pit, different crystallographic plane surface shows different contrast even if the angle of the primary electron incidence toward all those surfaces is the same. This is because of the acceptance of the signal detection system. In a present study we combined two electron trajectory simulations mentioned above and calculated electron trajectories both in and out of the specimen, to simulate the trajectory from the point of the signal generated until the signal is detected.

Although several simulation models of electron scatterings in a specimen have been reported to estimate the SE intensity at the surface, the model should be available to trace low energy (<50eV) electron trajectories. The model used here is basically the same as that reported in previous papers, and only a brief explanation is given in the following. Here, we made several assumptions as; [l]the energy loss of the primary and excited fast electrons is proportion to the number of SEs generated in the specimen, [2]the generated SE has an energy distribution as described by the Streitwolf equation, [3]the energy of the generated SEs are transferred to free electrons of the atom by the elastic-binary-collision, then one SE excited by the primary electron produces a ternary electron after the collision, and each one of the SE and the ternary electron produces higher order electrons in a cascade fashion. The simulation continues until the energy of each electron is less than the surface potential barrier. Angular and energy distributions and number of electrons emitted at the surface agree quite well with each experimental result in a typical case.
Object Restoration in 3D Electron Microscopy and Inverse Problems
Authors: Barth Martin
Published: June 2020

Standard 3D object reconstructions in electron microscope applications may be described as the solution of a forward problem: a series of projections is processed to collect reciprocal space data and a usually subsequent inverse Fourier transform yields a 3D objects representation. The resulting structure is commonly impaired by a convolution with an effective pointspread function that results fro an instrumental transfer function and the sampling character of the data.

Corresponding artificial features in the reconstruction may be reduced, if not eliminated, by approaching object restorations as solution of an inverse problem. In particular the problem with unevenly sampled data or "missing data" may considerably be alleviated.

With undersampled or unevenly sampled data the inverse problem tends to be ill-posed, i.e. the reconstruction as solution of the inverse problem tends to be non unique and sensitive to errors in the data such as noise. This ambiguity and sensitivity needs control by regularisation techniques. We discuss some general techniques such as projections onto convex sets (POCS), Maximum Entropy and Bayesian estimation with emphasis on applications to electron microscope data.
Detection, Averaging, and 3D Reconstruction of Biological Specimens on Hypercubes (Transputer-based) Computers
Authors: Carazo Jose-Maria , Benavides I. , Marco S. , Carrascosa J.L. , Zapata E.L.
Published: June 2020

Obtaining the three-dimensional (3D) structure of negatively stained biological specimens at a resolution of, typically, 2 - 4 nm is becoming a relatively common practice in an increasing number of laboratories. A combination of new conceptual approaches, new software tools, and faster computers have made this situation possible. However, all these 3D reconstruction processes are quite computer intensive, and the middle term future is full of suggestions entailing an even greater need of computing power. Up to now all published 3D reconstructions in this field have been performed on conventional (sequential) computers, but it is a fact that new parallel computer architectures represent the potential of order-of-magnitude increases in computing power and should, therefore, be considered for their possible application in the most computing intensive tasks.

We have studied both shared-memory-based computer architectures, like the BBN Butterfly, and local-memory-based architectures, mainly hypercubes implemented on transputers, where we have used the algorithmic mapping method proposed by Zapata el at. In this work we have developed the basic software tools needed to obtain a 3D reconstruction from non-crystalline specimens (“single particles”) using the so-called Random Conical Tilt Series Method. We start from a pair of images presenting the same field, first tilted (by ≃55°) and then untilted. It is then assumed that we can supply the system with the image of the particle we are looking for (ideally, a 2D average from a previous study) and with a matrix describing the geometrical relationships between the tilted and untilted fields (this step is now accomplished by interactively marking a few pairs of corresponding features in the two fields). From here on the 3D reconstruction process may be run automatically.
Angular Calibration of Ribosomal Subuntts in Factor Space
Authors: Pujari Minakhi , Frank Joachim
Published: June 2020

In single-particle analysis of macromolecule images with the electron microscope, variations of projections are often observed that can be attributed to the changes of the particle’s orientation on the specimen grid (“rocking”). In the multivariate statistical analysis (MSA) of such projections, a single factor is often found that expresses a large portion of these variations. Successful angle calibration of this “rocking factor” would mean that correct angles can be assigned to a large number of particles, thus facilitating three-dimensional reconstruction.

In a study to explore angle calibration in factor space, we used 40S ribosomal subunits, which are known to rock around an axis approximately coincident with their long axis. We analyzed micrographs of a field of these particles, taken with 20° tilt and without tilt, using the standard methods of alignment and MSA. The specimen was prepared with the double carbon-layer method, using uranyl acetate for negative staining. In the MSA analysis, the untilted-particle projections were used as active, the tilted-particle projections as inactive objects. Upon tilting, those particles whose rocking axes are parallel to the tilt axis will change their appearance in the same way as under the influence of rocking. Therefore, each vector, in factor space, joining a tilted and untilted projection of the same particle can be regarded as a local 20-degree calibration bar.
Feasibility of the Common-Axis Method for Alignment Prior to 3D Reconstruction in Dark-Held Electron Microscopy
Authors: Farrow Neil A , Peter Ottensmeyer F
Published: June 2020

Dark field electron microscopy has allowed the atomic modelling of the 3D structure of a number of biological molecules from images of single molecules and protein complexes. A prerequisite for such determinations has been knowledge as to the projection directions yielding the various images. The determination of these directions has relied on either the correlation of the dark field images with known biochemical data or with structures determined by x-ray crystallography. The use of information from such sources allows orientation of the different dark field images relative to each other. It will often be the case however that no other information is available as to the structure of the molecule under investigation. It then becomes necessary to use the images alone to determine their orientation relative to each other prior to using 3D reconstruction techniques.

A number of methods are available to determine projection directions from a set of images of a given object. Most notable among these methods are the method of moments and the common axis method. The common axis method uses the fact that any two views of the same object share a common line projection. Thus by comparing line projections of three images it is possible to orientate these images relative to each other in space.
Method for Reduction of Quantum Noise Recorded on Imaging Plate at Low Dosage
Authors: Ishikawa Arira , Suda Hitoshi , Fukami Akira , Oikawa Tetsuo , Arai Yoshihiro
Published: June 2020

To record radiation sensitive specimens at low dosage, a recording media with high sensitivity is required. Conventionally, some x-ray films have been used for this purpose. According to our experiences, however, its own granular noise is so severe that the practical sensitivity is restricted to the order of 10-13 C/cm2. The imaging plate (IP), recently developed for a transmission electron microscope, is a promising recording media, as it has a wide dynamic range, good linearity and high sensitivity of the order of 1×10-14 C/cm2 for the electron beam. The grain size of photostimulable phosphor, which composes the phosphor layer in the IP, is about 5 μm. As image recorded in the IP is read out as the digital image with the pixel size of 50 μm, the granularity of the IP can be negligible. The increase of the quantum noise, due to the fluctuation of the number of electrons per each pixel, at the low dosage will restrict the practical sensitivity of the IP. In order to raise this sensitivity, we applied the filtering technique which was developed for reduction of the granular noise of high sensitive x-ray film.
The Contribution of Intermediate-Voltage, High-Resolution Electron Microscopy In Materials Science: An Appraisal
Authors: Hutchison John L.
Published: June 2020

Over the past five years or so the development of a new generation of high resolution electron microscopes operating routinely in the 300-400 kilovolt range has produced a dramatic increase in resolution, to around 1.6 Å for “structure resolution” and approaching 1.2 Å for information limits. With a large number of such instruments now in operation it is timely to assess their impact in the various areas of materials science where they are now being used. Are they falling short of the early expectations? Generally, the manufacturers’ claims regarding resolution are being met, but one unexpected factor which has emerged is the extreme sensitivity of these instruments to both floor-borne and acoustic vibrations. Successful measures to counteract these disturbances may require the use of special anti-vibration blocks, or even simple oil-filled dampers together with springs, with heavy curtaining around the microscope room to reduce noise levels. In assessing performance levels, optical diffraction analysis is becoming the accepted method, with rotational averaging useful for obtaining a good measure of information limits. It is worth noting here that microscope alignment becomes very critical for the highest resolution.

In attempting an appraisal of the contributions of intermediate voltage HREMs to materials science we will outline a few of the areas where they are most widely used. These include semiconductors, oxides, and small metal particles, in addition to metals and minerals.
Microanalysis At Intermediate Voltages
Authors: Williams David B. , Michael Zemyan S.
Published: June 2020

Microanalysis using x-ray emission spectrometry (XES) or electron energy-loss spectrometry (EELS) at intermediate voltages should offer advantages over 100kV instruments.  Brightness increases linearly with kV (for thermionic sources), but the inelastic scattering cross section decreases.  Potential improvements in detectability limit and spatial resolution in both XES and EELS have been major factors spurring the development of intermediate voltage AEMs.

The x-ray peak to background ratio increases with kV (Figure 1), and improvements in detectability limits have been reported at 300kV. The variation of detectability with spatial resolution is discussed elsewhere in these proceedings. Beam spreading should decrease linearly with kV, but experimentally the spatial resolution (Figure 2a,2b) does not show the expected improvement. This may be due to increased beam broadening from fast secondary electrons. The smallest probe size consistent with generating sufficient signal (i.e. an FEG) is better than increased kV for improved spatial resolution3. Increased kV has seen the return of the ‘hole count’ problem in XES. Higher voltage electrons generate harder x-rays at the C aperture, and current aperture design cannot sufficiently restrict the xray flux. The flux at 300kV can contribute up to 10% of the characteristic x-ray signal, which severely compromises the microanalysis quality. An FEG with reduced source size may help. At intermediate voltages, intrinsic Ge (IG) detectors can be used for analysis of Kα, lines from high atomic number elements. K line analysis gives improved accuracy compared with L and M line analysis for which Cliff- Lorimer k factors, (both experimental and theoretical) are extremely variable, possibly due to fast secondaries also. IG detectors can detect Au K lines, resolve Kα1Kα2 lines, (see Figure 3) offer improved energy resolution (~120eV) over Si(Li) detectors, and may be more stable under electron irradiation. Continued improvement in IG detectors may result in their displacing Si(Li) detectors from most AEMs.
High-resolution Study of DNA in Vitrified Sections
Authors: Richter Karsten , Dubochet Jacques
Published: June 2020

Water in biological specimens, when cooled at a very high rate can be immobilized in a metastable, amorphous, so-called vitreous state. Kept vitreous at temperatures below 130 K, the vapour presure of water is so low, that the specimens will not dry when observed directly in the EM. Therefore, cryo-EM of vitrified biological specimens ommits artifacts due to dehydration and chemical fixation.

One field of interest in the application of this technique is the study of the structural organisation of genomic material in situ.

The objects to be studied, i.e. isolated nuclei, culture cells, or tissues are all too large to allow investigation as a whole in the TEM; they must be observed as ultrathin sections. While the cutting of vitrified bulk specimens into ultrathin sections is possible, the cutting process provokes severe deformation: The sections are compressed along the cutting direction by ca 50% and correspondingly, the section thickness doubles in respect to the actual advance of the microtome, which indicates important displacements of material takeing place during sectioning. Nevertheless structural information even in small range order can be conserved.
Structure of The Z Disk of Sectioned Bee Flight Muscle
Authors: Deatherage J.F. , Cheng N.
Published: June 2020

Information from oblique thin sections and from 3-D reconstructions of tilted transverse thin sections has been combined to determine the 3-D structure of the honeybee flight muscle Z disk at 7øÅ resolution. The overall symmetry and structure of the Z disk and its relationship to the rest of the myofibril have been determined by tracing filaments and connecting elements on electron images of oblique sections which have been enhanced by a local crystallographic averaging technique .

The 3-D structure of the central region of the Z disk of honeybee flight muscle has been determined by crystallographic 3-D reconstruction from electron micrographs of tilted thin sections. The reconstructions show a complex network in which actin filaments terminate and are crosslinked together (Figure 1); a number of structural domains are resolved in the network.

The central region of the Z disk contains two sets of overlapping actin filaments of opposite polarity, which originate in the sarcomeres adjacent to the Z disk, and connections between these filaments. The filaments are deflected by the attachment of crosslinks; spacings between filaments change by over 1øøÅ during their passage through the Z disk. Each actin filament is linked by connecting structures to four filaments of opposite polarity and two filaments of the same polarity.
Electron Microscope Tomography (Emt): Reconstruction Algorithms, Imaging and Mass Loss of Thick Sections
Authors: Olins A.L. , Levy H. A. , Shah M. B. , Lamprecht G. G. , Olins D. E.
Published: June 2020

We previously reported EMT reconstructions of thick (>0.5 μm) sections using electron spectroscopic imaging at the most probable electron energy loss (ΔEp). We2 calculated values of contrast and signal-to-noise ratio (SNR) for images formed with electrons taken throughout the spectrum. We observed maximal SNR just below ΔEp, and found that changes in the electron beam voltage and mass thickness (t/λ) led to predictable changes in the EELS.

We have begun a study of mass loss due to electron exposure of thick epon sections. Uranyl and lead stained sections were put on formvar covered grids and carbon coated on each side. Clear epon regions were analyzed. During each exposure series the beam was kept at crossover; the beam current was changed from its lowest value (0.5 μamp) to its highest value (55 μamp). As reported elsewhere, mass loss occurs early and plateaus. Calculations were based on EELS collected at different times during each series (Figure 1a). Measured positions of ΔEp at different times (min) of maximum beam current irradiation are shown in Figure 1 b. Mass thickness, t/λ = In (total area/zero loss area), shows a linear relationship with ΔEp (Figure 1c). Using λ = 115 nm in epon, we calculate the initial thickness of the sections to be 0.5, 0.75, 1.0 μm and the mass loss to be ∼37 nm. If mass loss is independent of the specimen thickness, then the 50% loss observed with thinner specimen becomes a trivial 4-7% loss in sections of 0.5 to 1.0 μm.