Journal of electron microscopy technique最新文献

High-voltage electron microscopy has shown itself advantageous for the study of natural science, including biology, but especially for materials science. The most important advantage for materials science is for in situ experiments about the detailed processes of the phenomena that occur in bulk materials. The present paper is mainly concerned with several types of in situ experiments that have been carried out in the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University. The following subjects have been studied: a) fundamental problems, such as the conditions necessary for in situ experiments, functional features of specimen treatment devices, and the effects of electron irradiation; b) the dislocation behavior of crystals under various conditions; c) high-temperature behavior of refractory materials, mainly ceramic composites; d) new applications of electron irradiation effects, such as amorphization of crystalline materials and electron-irradiation-induced foreign-atom implantation; e) environment-matter interaction, mainly chemical amorphization of alloys; and f) future trends of the in situ experiment, such as combinations with Auger valency electron spectroscopy and high-resolution electron microscopy.

Thermionic emission properties of several kinds of refractory carbides, nitrides, and borides of the transition metals in the form of powder were investigated with a newly developed measuring device and evaluated by the figure of merit defined as the ratio of the effective work function to the working temperature at which the vapor pressure becomes 1 x 10(-5) Torr. There are several materials whose thermionic emission properties are better than those of tungsten or compatible to those of tungsten among the carbides and borides, such as TaC, HfC, ZrC, LaB6, and CeB6, as judged by the figure of merit. New preparation methods for carburization, nitriding, and boriding of the wires of matrix metals and alloys were successfully developed for using these materials as the cathode of the electron microscope. Other necessary techniques such as spot welding and electrolytic etching were also developed. From the brightness characteristics, it was found that some of carbides, carbide solid solutions, and borides such as HfC, ZrC (Ta0.8-0.7Hf0.2-0.3)C, TaB2, and HfB2 are very good emitters comparable to LaB6. It is emphasized that the work functions of the carbide-solid solutions (Ta0.8Hf0.2)C and (Ta0.7Hf0.3)C, which have low rates of evaporation at high temperature, show no remarkable rise as compared with that of HfC, so that their figures of merit are better than that of HfC. Feasibility of providing good cathodes with HfC and (Ta0.8Hf0.2)C tips was demonstrated by taking high-resolution electron micrographs.

Four kinds of works on the detection of displacement of atoms in crystals are shown. The irregular small displacement of atoms has been detected, with an accuracy of about 0.1 A around dislocations and stacking faults in Au crystals as shown by their electron microscope images. The displacement of the atomic images is recorded by aberration-free focus (AFF). Even when the periodic displacement of atoms in SiC and TaS2 crystals is around 0.1% of the lattice constant, this displacement has been revealed as the weak-contrast anomaly in the images. Using in situ observations by a TV system attached to an electron microscope, the rapid movements of atoms that have taken place within 1/30 sec have been recorded. Using the technique of successive subtraction of the images by TV system and image sigma, only the images of moving atoms in Au crystal have been recorded each 1/10 sec.

Application of electron microscopy in a wide variety of fields of investigation has placed ever-expanding demands on the various components of the instrument. In situ specimen manipulation is one such demand and can often be critical to the success of an experiment. Control of specimen orientation is the most common manipulation, but control of a variety of other physical and chemical parameters may also be important. Temperature, gaseous and/or liquid environment, and mechanical operations are examples. Control and variation of these parameters in a small device (occupying a few cm3) operated in a strong magnetic field inside a vacuum system is often a considerable challenge. This must also be done at extreme stability: at least as good as the resolution limit of the microscope. Optimization of stage performance is too often sacrificed for optical performance or vice versa. The next generation of objective lenses and specimen stages are being designed in concert: an approach which should lead to an improved in situ laboratory, observed with optimum optics.

Silver enhancement of immunogold-labeled cells was carried out to increase the applicability of colloidal gold probes for visualization in the backscatter electron imaging (BEI) mode of a scanning electron microscope. Optimum conditions were established for single particle discrimination and differential counting of labeling density at low magnifications. Red blood cells double-labeled with 15 + 40 nm and 5 + 20 nm gold probes were silver-enhanced for 6 min and 20 min, respectively, at which times both pairs of labels increased to about 25 + 50 nm. The gold probes still appeared spherical after enhancement and were easily discriminated. Cells were also single-labeled with the above probes and enhanced accordingly. The present method enables visualization of individual particles of any probe size, labeling one, or simultaneously two, antigenic sites on cell surfaces. The silver enhancement procedure thereby allows cells to be labeled with small probes with increased labeling efficiency.

An image can be represented digitally as a matrix of numbers. When those numbers are linearly related to a property of the object, such as mass per unit area, a simple integration of an image area leads to a total of that property, such as the mass of a particle that is represented in a selected area. Following techniques pioneered by Bahr and Zeitler, we illustrate the use of photographic densitometry of films exposed in an electron microscope to measure electron scattering. The transmission of an electron micrograph will be linear with respect to mass thickness for a particular value of background brightfield density, hence allowing determination of the mass of microscopic particles. We show here a digital computer method for conveniently establishing the linear condition by quantitative image processing using micrographs of polystyrene spheres. The method also serves to produce calibration curves for cases where the transfer from transmission to mass thickness is not linear. We also illustrate how an inexpensive computer is used to display and integrate regions of micrographs to determine particle mass.

A comprehensive computer-graphics-based system (STERECON) is described for tracing and digitizing contours from individual or stereopair electron micrographs. The contours are drawn in parallel planes within the micrographs. Provision is also made for tracing and digitizing in full three-dimensional (3-D) coordinates in any direction along linear structures such as cytoskeletal elements. The stereopair micrographs are viewed in combination with the contours being traced on a graphics terminal monitor. This is done either by projecting original electron micrograph (EM) negatives onto a screen and optically combining these images with contour lines being drawn on the monitor, or by first digitizing the images and displaying them directly on the monitor along with the contour lines. Prior image digitization allows computer enhancement of the structures to be contoured. Correction and alignment routines are included to deal with variable section thickness, section distortion and mass loss, variations in photography in the electron microscope, and terminal screen curvature when combining projected images with contour lines on the monitor. The STERECON system organizes and displays the digitized data from successive sections as a 3-D reconstruction. Reconstructions can be viewed in any orientation as contour stacks with hidden lines removed; as wire-frame models; or as shaded, solid models with variable lighting, transparency, and reflectivity. Volumes and surface areas of the reconstructed objects can be determined. Particular attention was paid to making the system convenient for the biological user. Users are given a choice of three different stereo-viewing methods.

To overcome the radiation damage-induced limitations to the resolution of three-dimensional reconstructions from electron microscopic tilt series, novel reconstruction schemes have been developed that require only a single exposure of the specimen. The tilt series collected with these methods have random projection directions. First, three-dimensional reconstruction techniques are described that are applicable to data obtained from tilt series with regular tilt geometry, followed by the extensions of these techniques to permit analysis of projection series with randomly spaced tilts. The main emphasis is placed on the weighted back-projection methods, which have recently been extended so as to be applicable to random tilt series. Besides a description of the algorithms, the complete procedure for a three-dimensional reconstruction from a single-exposure, random conical tilt series is explained, including the determination of the azimuthal angles, the alignment scheme for conical tilt series, the dependence of the achievable resolution on the number of projections for regular conical and single-axis geometries, and the method to calculate the actual resolution of two-dimensional image averages and of three-dimensional reconstructions using the phase residual and Fourier ring correlation criteria. Examples are given of biological specimens to which these three-dimensional reconstruction methods have been applied.

Methods are described for the analysis of electron micrographs of biological objects with helical symmetry and for the production of three-dimensional models of these structures using computer image reconstruction methods. Fourier-based processing of one- and two-dimensionally ordered planar arrays is described by way of introduction, before analysing the special properties of helices and their transforms. Conceiving helical objects as a sum of helical waves (analogous to the sum of planar waves used to describe a planar crystal) is shown to facilitate analysis and enable three-dimensional models to be produced, often from a single view of the object. The corresponding Fourier transform of such a sum of helical waves consists of a sum of Bessel function terms along layer lines. Special problems deriving from the overlapping along layer lines of terms of different Bessel order are discussed, and methods to separate these terms, based on analysing a number of different azimuthal views of the object by least squares, are described. Corrections to alleviate many technical and specimen-related problems are discussed in conjunction with a consideration of the computer methods used to actually process an image. A range of examples of helical objects, including viruses, microtubules, flagella, actin, and myosin filaments, are discussed to illustrate the range of problems that can be addressed by computer reconstruction methods.