Modern Electron Microscopes Research Articles

Horseradish peroxidase cDNA as a marker for electron microscopy in neurons

A reliable and convenient marker of gene transfer in neurons is lacking for electron microscopy. To facilitate the use of molecular genetic approaches in such studies, we introduce the use of horseradish peroxidase (HRP) cDNA as a marker that identifies transfected neurons for correlated light and electron microscopy (EM). By flanking the cDNA with an endoplasmic reticulum (ER)-retention signal, expressed HRP was localized and retained inside the ER of neurons. The construct was placed under the control of the synapsin promoter resulting in neuron-specific expression of HRP. After transfection of hippocampal neurons in culture and visualization of HRP with diaminobenzidine (DAB) after fixation, both cell bodies and proximal dendrites of HRP-positive neurons were readily identifiable by light microscopy. After processing for EM, the heavily DAB-labeled ER was obvious in both cell bodies and dendrites of transfected neurons. In addition, the labeling of distal dendrites, axons, and synapses was apparent. The structural preservation of transfected neurons was optimal as is typical for glutaraldehyde-fixed tissue. Due to the ER-retention signal, HRP was contained inside the ER and the ultrastructure of all other components was not occluded and was quantitatively accessible. These characteristics make HRP co-transfection a potentially powerful tool for modern electron microscopy in both cell biology and neuroscience.

Determination of the chiral indices (n,m) of carbon nanotubes by electron diffraction

The atomic structure of a carbon nanotube can be defined by the chiral indices, (n,m), that specify its perimeter vector (chiral vector), with which the diameter and helicity are also determined. The fine electron beam available in a modern Transmission Electron Microscope (TEM) offers a unique and powerful probe to reveal the atomic structure of individual nanotubes. This article covers two aspects related to the use of the electron probe in the TEM for the study of carbon nanotubes: (i) to express the electron diffraction intensity distribution in the electron diffraction patterns of carbon nanotubes and (ii) to obtain the chiral indices (n,m) of carbon nanotubes from their electron diffraction patterns. For a nanotube of given chiral indices (n,m), the electron scattering amplitude from the carbon nanotube can be expressed analytically in closed form using the helical diffraction theory, from which its electron diffraction pattern can be calculated and understood. The reverse problem, i.e., assignment of the chiral indices (n,m) of a carbon nanotube from its electron diffraction pattern, is approached from the relationship between the electron diffraction intensity distribution and the chiral indices (n,m). The first method is to obtain indiscriminately the chiral indices (n,m) by reading directly the intensity distribution on the three principal layer lines, l(1), l(2), and l(3), which have intensities proportional to the square of the Bessel functions of orders m, n, and n + m: I(l1) proportional, variant |J(m) (pidR)|(2), I(l2) proportional, variant |J(n) (pidR)|(2), and I(l3) proportional, variant |J(n+m) (pidR)|(2). The second method is to obtain and use the ratio of the indices n/m = (2D(1)-D(2))/(2D(2)-D(1)) in which D(1) and D(2) are the spacings of principal layer lines l(1) and l(2), respectively. Examples of using these methods are also illustrated in the determination of chiral indices of isolated individual single-walled carbon nanotubes, a bundle of single-walled carbon nanotubes, and multi-walled carbon nanotubes.

Low energy focused ion beam system design

Focused ion beam (FIB) systems operated at typically 30keV energy have now reached a remarkable level of sophistication in terms of optical performance. Not unlike the much earlier developments in scanning electron microscopy, there now seems to be a fairly recent trend towards also enhancing the low energy performance of these systems—mainly driven by their possible utilization in transmission electron microscopy sample preparation. However, low energy operation indeed appears to have already been an issue in the very early days of FIB development—although then against the background of potential applications in direct semiconductor device fabrication. In this study, different approaches for generating low energy ion probes are discussed on the basis of previous and existing FIB systems for which experimental data have been made available. Subsequently, a dedicated low energy focused ion beam column design is introduced, which operates similarly to many modern field emission scanning electron microscopes. The performance of the system is assessed by means of standard aberration-optical calculations as well as by using data from direct ray tracing. The latter also allows the consideration of the impact of mutual Coulomb interactions.

Probing carrier dynamics in nanostructures by picosecond cathodoluminescence

Picosecond and femtosecond spectroscopy allow the detailed study of carrier dynamics in nanostructured materials. In such experiments, a laser pulse normally excites several nanostructures at once. However, spectroscopic information may also be acquired using pulses from an electron beam in a modern electron microscope, exploiting a phenomenon called cathodoluminescence. This approach offers several advantages. The multimode imaging capabilities of the electron microscope enable the correlation of optical properties (via cathodoluminescence) with surface morphology (secondary electron mode) at the nanometre scale. The broad energy range of the electrons can excite wide-bandgap materials, such as diamond- or gallium-nitride-based structures that are not easily excited by conventional optical means. But perhaps most intriguingly, the small beam can probe a single selected nanostructure. Here we apply an original time-resolved cathodoluminescence set-up to describe carrier dynamics within single gallium-arsenide-based pyramidal nanostructures with a time resolution of 10 picoseconds and a spatial resolution of 50 nanometres. The behaviour of such charge carriers could be useful for evaluating elementary components in quantum computers, optical quantum gates or single photon sources for quantum cryptography.

Effects of electron irradiation in nuclear waste glasses

This article summarizes recent studies of electron irradiation damage in sodium borosilicate, iron phosphate and aluminophosphate glass waste forms using a modern analytical electron microscope. Three different borosilicate (wt%) (17.72% B2O3–16.67% Na2O–64.61% SiO2, 17.78% B2O3–15.83% Na2O–61.39% SiO2–4.99% Fe2O3 and 17.86% B2O3–15.90% Na2O–61.63% SiO2–4.61% FeO) and iron phosphate (mol%) (45% Fe2O3–55% P2O5, 20% Fe2O3–80% P2O5, and 20% Fe2O3–20% Na2O–60% P2O5) glasses, and an aluminophosphate (mol%) (44.5% P2O5–31.5% Al2O3–20.2% Na2O–3.8% K2O) glass were studied. Results indicate that all these glasses decomposed under the 200 kV electron irradiation (at doses higher than 1.0 × 1026 e m−2). Migration of alkali elements from the irradiated centres to the peripheries under irradiation occurred in the alkali element-containing glasses, which results in the formation of alkali element-depleted and -enriched phases. Formation of bubbles was only observed in the alkali element-containing iron phosphate and aluminophosphate glasses, not in sodium borosilicate glasses when irradiated over a broad of dose rates (1.6 × 1022 e m−2 s to 8 × 1026 e m−2 s). Separation of boron-rich phase from silicon-rich phase, iron-rich/aluminium-rich phase from phosphorous-rich domains were observed in the three types of glasses, respectively. Further irradiation resulted in formation of small particles. In Fe-containing borosilicate glasses, the Fe is associated with the boron-rich phases after phase separation.

Electron microscope calibration for the Lorentz mode

The calibration of a modern electron microscope for Lorentz microscopy observations has been performed using diffractogram, Fresnel diffraction fringe analysis and low-angle electron diffraction methods. An example related to the observations of electrostatic fields associated to a thinned reverse-biased p–n junction is also reported.

Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.

High-resolution transmission electron microscopy: the ultimate nanoanalytical technique.

To be able to determine the elemental composition and morphology of individual nanoparticles consisting of no more than a dozen or so atoms that weigh a few zeptograms (10(-21) g) is but one of the attainments of modern electron microscopy. With slightly larger specimens (embracing a few unit cells of the structure) their symmetry, crystallographic phase, unit-cell dimension, chemical composition and often the valence state (from parallel electron spectroscopic measurements) of the constituent atoms may also be determined using a scanning beam of electrons of ca. 0.5 nm diameter. Nowadays electron crystallography, which treats the digital data of electron diffraction (ED) and high-resolution transmission electron microscope (HRTEM) images of minute (ca. 10(-18)g) specimens in a quantitatively rigorous manner, solves hitherto unknown structures just as X-ray diffraction does with bulk single crystals. In addition, electron tomography (see cover photograph and its animation) enables a three-dimensional picture of the internal structure of minute objects, such as nanocatalysts in a single pore, as well as structural faults such as micro-fissures, to be constructed with a resolution of 1 nm from an angular series of two-dimensional (projected) images. Very recently (since this article was first written) a new meaning has been given to electron crystallography as a result of the spatio-temporal resolution of surface phenomena achieved on a femtosecond timescale.

Atom Probes Leap Ahead

Abstract Since early times, the collective understanding of our microscopic universe has been directly tied to the quality of our microscopies. This has been true from the advent of light microscopes through to modern electron microscopes. Indeed, if one is to work on a given scale, one must be able to “see” at that scale. At the beginning of the 21st century, human inquiry is focused on the atomic scale.

A Quantitative Nanodiffraction System for Ultrahigh Vacuum Scanning Transmission Electron Microscopy

Of all the long-lived particles available as probes of condensed matter, and of all the signals available on a modern electron microscope, electron nanodiffraction patterns provide the strongest signal from the smallest volume. The technique is therefore perfectly suited to nanostructural investigations in inorganic chemistry and materials science. The Vacuum Generators HB501S, an ultrahigh vacuum (UHV) variant of the HB501 scanning transmission electron microscope (STEM), with side-entry double-tilt stage, specimen preparation and analysis chamber, three postspecimen lenses, and cold field-emission tip with integral magnetic gun lens, has therefore been modified to optimize nanodiffraction and quantitative convergent beam electron diffraction (QCBED) performance. A one-micrometer grain-size phosphor screen lying on a fiber-optic faceplate atop the instrument is fiber-optically coupled to a 2048 x 2048 charge-coupled device (CCD), 16-bit camera. This arrangement promises to provide much greater sensitivity, larger dynamic range, and a better modulation transfer function (MTF) than conventional single crystal scintillator (YAG) CCD systems, with noticeable absence of cross talk between pixels. The design of the nanodiffraction detector system is discussed, the gain of the detector is measured, the spherical aberration constant of the objective lens is measured by the Ronchigram method, and preliminary results from the modified instrument are shown.

Quantitative Evaluation of Focal Series in HRTEM

Due to the fact that the information limit of modern mid-voltage high-resolution electron microscopes reaches or even surpasses the 1 Angstrom limit, the precise control of the objective lens aberrations becomes more and more crucial in order to obtain directly interpretable results. Such lens aberrations must be either hardware-corrected before the experiment or can be determined and removed after the experiment by software in the case of phase-retrieval methods. Apart from the frequently discussed anisotropic residual aberrations like coma, twoand three-fold astigmatism, also the allowed tolerance in the classical isotropic defocus aberration has to be reconsidered critically when approaching the 1 Angstrom limit.

Reconstruction of the Projected Potential from a through Voltage Series of Dynamical Electron Diffraction Patterns Including Absorption

Recently several different methods have been proposed to reconstruct the projected crystal potential from electron diffraction patterns. These methods envolve either diffraction patterns at many different orientations (as many orientations as beams in the pattern) and/or images, which makes their experimental realization difficult. We propose an entirely new method for reconstructing the projected crystal potential from fully dynamical [including absorption and multiple scattering effects to all orders] diffraction patterns from only a single crystal orientation and no image at all. Knowledge of the specimen thickness is not necessary. However, it requires diffraction patterns at many different accelarating voltages, which is a parameter that can easily be varied (within a certain range) in most modern electron microscopes. Since the intensities in the electron diffraction pattern are not affected by lens abberations this method is capable of reconstructing the projected potential with a resolution far better than that of any method using HRTEM images.

Grain Boundary Structure Analysis of Covalent Bonding Materials by Atomic Resolution Transmission Electron Microscopy

Abstract Grain boundaries of covalent bonding materials such as silicon, germanium and SiC require rather physical consideration than that of geometry to discuss the result of their electron microscopic observation. Dangling bond, for example, is expected least in number in the stable boundary (least dangling bond rule). [1][2] in practice, further precise consideration must be done both in geometry and in physics in the covalent bonding materials boundary. in the present study, geometrical discussion is combined together with that of the atomic bonding about diamond, SiC and ZnO grain boundaries. Chemical structure image of the modern high-voltage transmission electron microscope enabled to do it. An asymmetrical Σ3 boundary of diamond and a 6H-SiC boundary with 70.5 degree rotation are mainly observed. The sliced specimen, 0.3mm in thickness, was mechanically thinned down to 0.02 mm and was finally polished using an argon ion milling. Final thickness of the specimen was estimated to be 3 nm in the region close to the edge of the small hole.

The Triebenberg Laboratory-Designed for Highest Resolution Electron Microscopy and Holography

Abstract Very often, the performance of modern electron microscopes is limited by the poor quality of the laboratory rather than by the specifications of the microscope. Therefore, with microscopes aiming at subangstrom resolution microscopy and highest performing holography methods, special measures have to be taken to reach the needed low level of AC-stray fields, vibrations, and acoustic noise. in an ”organically“ grown institute, this is very difficult to realize, if possible at all. • The Triebenberg Lab is newly built well outside the city of Dresden, in an area essentially free from disturbances, i.e. free from power lines, away from roads, trains and airplanes; fortunately, the site is shielded from wind by means of a dense belt of trees. The lab houses six microscope units each consisting of a microscope room, a room for peripheral devices (power supply, cooling units, computers, etc), and an office for up to three scientists. in view of the nearly ideal environment, the main challenge is to avoid any self-made disturbances which might arise by the operation of the lab. Therefore, without any compromise using the best art of engineering, we designed a special lab virtually free from disturbances. in the following, the main guidelines for the construction are sketched.

Structural change of amorphous Fe 90Zr 7B 3 alloy in the primary crystallization process studied by modern electron microscope techniques

Structural changes in the primary crystallization process of amorphous Fe 90Zr 7B 3 alloy have been investigated using modern electron microscope techniques. In the as-quenched specimen, bcc–Fe clusters as small as 1 nm were observed by high resolution electron microscopy (HREM). Nanoprobe energy dispersive X-ray spectroscopy revealed a development of Zr-enriched zones beside the α-Fe nanocrystals in the initial crystallization stage at 733 K. Besides the α-Fe nanocrystals, Fe 2Zr and Fe 3Zr-type nanoprecipitates were identified by nanodiffraction and HREM after annealing at 923 K. Electron diffraction pair distribution function (PDF) analysis were performed for the as-quenched and annealed specimens. The PDF analysis of the amorphous matrix combined with the reverse-Monte-Carlo simulation revealed that bcc-like Fe clusters are formed already in the as-quenched state. The coordination number and Zr–Fe distance of the short-range order (SRO) structures around Zr atoms in the amorphous matrix both decreased on annealing towards a formation of local structures resembling structure units in the Fe 2Zr or Fe 3Zr phases. The PDF and HREM results strongly suggest a polymorphic reaction from the Zr-enriched matrix regions to the Fe 2Zr or Fe 3Zr nanoprecipitates in the crystallization stage. The formation of the Fe 2Zr or Fe 3Zr-like SROs in the amorphous matrix is thought to stabilize the matrix structure, and contributes to suppress the growth of α-Fe nanocrystals towards the optimized structure of the good soft-magnetic property.

Analysis of prehispanic pigments from “Templo Mayor” of Mexico city

The application of modern electron microscopy and X-ray diffraction techniques to the study of ancient pigments has proved to be very useful. In the present work we report the study of pigments from Mexica (Aztec) culture which developed in Central Mexico from 1325–1521 AD; we study the blue, ochre, red and black pigments. We found in the most cases the paints were made of a clay substrate that contained metal particles such as Fe, Ti, Mn and Zn, in different phases. This technology was found similar to that of earlier mesoamerican cultures.

Probability calculus for quantitative HREM. Part I: Monte-Carlo and point cloud techniques

A new approach to a central question of modern high-resolution electron microscopy (HREM) is presented: How precisely can we locate atom positions in crystal defects using computer-controlled structure retrieval algorithms? The purpose is not just to give error bars for the determined atomic column positions, but to derive estimations for the continuous probability functions. In the first part of this two-part paper, we present techniques which analyse point clouds of fluctuating fit-results for atom coordinates. The point clouds are obtained in a first approach from multiple input images differing in noise, commonly known as Monte-Carlo error estimation. Furthermore, we exploit the response obtained during a global optimisation-based refinement process for which all the trial structures are evaluated resulting in a second type of point cloud. In comparison, the Monte-Carlo-type technique turns out to be the most robust one. Using examples from current research on SrTiO 3-bicrystals and Cu–Al 2O 3 interfaces, we study two largely different crystallographic and statistical situations.

Tutorial: Tem Specimen Preparation in the Physical Sciencestripod Polishing and Ion Milling

Over the past few decades, the demands of modern analytical electron microscopy have increased the need for TEM specimen preparation techniques with a minimum of misleading artifacts in terms of chemical microanalysis. At the same time, the demands of modern industrial materials, be they semiconductor, polymeric or composite in nature, call for speed, flexibility and high spatial resolution as well. The response from the electron microscopy community, especially that portion in the private sector, have been to devise (or advocate) radically different forms of TEM thin specimen preparation from that of classic replication, electropolishing and ion thinning.This tutorial sets forth the goals of TEM specimen preparation, and the requirements for a "good" TEM specimen. The strategic choices governing which technique to use for preparing a wide variety of specimens will be covered. A TEM Specimen Preparation Flow Chart will be used to plot a course that makes optimum use of the preparation techniques available as a function of the type of specimen to be prepared.

Identification of Spherical Virus Particles in Digitized Images of Entire Electron Micrographs

New methods are described that should facilitate high-resolution (5–10 Å) image reconstructions from low-dose, low-contrast electron micrographs of frozen-hydrated specimens and processing of large, digital images produced by new imaging devices and modern electron microscopes. Existing techniques for automatic selection of images of individual biological macromolecules from electron micrographs are inefficient or unreliable. We describe the Crosspoint method (CP), which produces good quality solutions with relatively small miss rates and few false hits, and an extension of this method along with a procedure for refining its solution. Two algorithms for processing large images, one based on image subsampling, the other on image decomposition, are described. A large image is first compressed (e.g., by subsampling) and the CP method is applied to the compressed image to produce an initial solution. The information gathered at this stage is used to cut the original image into subimages and then to refine the particle coordinates in each subimage. An interactive environment for experimenting with particle identification methods is described.

Correction of three-fold astigmatism for ultra-high-resolution TEM

Due to the influence of three-fold astigmatism, the spatial resolution obtained on many modern high-resolution transmission electron microscopes is often not better than 0.2 nm. Although beam tilt can mask the effect of three-fold astigmatism when imaging periodic specimens, the correction of three-fold astigmatism is required for obtaining isotropic ultra-high resolution (≤0.15 nm). Three-fold astigmatism also hampers the correct alignment of the microscope, resulting in additional beam tilt and two-fold astigmatism, and consequently in a further loss of resolution. The amount of three-fold astigmatism of an instrument is found to be stable for periods well in excess of a year. After hardware correction through excitation of a magnetic hexapole in the objective lens stigmator unit of our Philips CM300ST FEG, we reduced the magnitude of the three-fold astigmatism from a value of 1.1 μm, for this particular instrument, to less than 0.1 μm, a value at which it allows at least 0.1-nm spatial resolution. Additionally, we show that the micrscope can now be properly aligned for ultra-high-resolution experiments using the standard manual alignment procedures.