A recent article in these pages compares STEM images with an image obtained with the One-Ångstrom Microscope (OÅM) at Lawrence Berkeley National Laboratory (LBNL). Although the experimental work is of excellent quality, Diebold et al. (2003) offer an incorrect explanation of the image formation process in the high-resolution transmission electron microscope. It is important that this misinterpretation be corrected before it comes to be accepted as factual by other scientists who are not expert in the field of high-resolution transmission electron microscopy.

The main purpose of the article by A.C. Diebold and coworkers (2003) is to propose a robust method for determination of gate oxide thickness. O'Keefe objects to a statement in this paper that “Lattice images do NOT depict the projected atom columns; instead, they are interference patterns of the directly transmitted beam with diffracted beams.”

Improvements in instrumentation and image processing techniques mean that methods involving reconstruction of focal or beam-tilt series of images are now realizing the promise they have long offered. This indirect approach recovers both the phase and the modulus of the specimen exit plane wave function and can extend the interpretable resolution. However, such reconstructions require the a posteriori determination of the objective lens aberrations, including the actual beam tilt, defocus, and twofold and threefold astigmatism. In this review, we outline the theory behind exit plane wavefunction reconstruction and describe methods for the accurate and automated determination of the required coefficients of the wave aberration function. Finally, recent applications of indirect reconstruction in the structural analysis of complex oxides are presented.

This article presents my reminiscences of the work in Chicago on the correction or reduction of lens aberrations. Studies began in the early 1960s and extended over a period of almost 40 years, although it was never the primary focus of the work of the laboratory. The account is almost entirely based on my own memory, which is not a very reliable instrument. It is not intended to be a review and is more accurately describable as a personal recollection.

During in situ transmission electron microscopy (TEM) field emission experiments, carbon nanotubes are observed to strongly diffract the imaging TEM electron beam. We demonstrate that this effect is identical to that of a standard electrostatic biprism. We also demonstrate that the nanotube biprism can be used to capture electron-holographic information.

A general method is presented for determining and correcting nonlinear position detector responses in single particle tracking as used in three-dimensional scanning probe microscopy based on optical tweezers. The method uses locally calculated mean square displacements of a Brownian particle to detect spatial changes in the sensitivity of the detector. The method is applied to an optical tweezers setup, where the position fluctuations of a microsphere within the optical trap are measured by an interferometric detection scheme with nanometer precision and microsecond temporal resolution. Detector sensitivity profiles were measured at arbitrary positions in solution with a resolution of approximately 6 nm and 20 nm in the lateral and axial directions, respectively. Local detector sensitivities are used to reconstruct the real positions of the particle from the measured position signals.

A technique is proposed for reducing unwanted diffraction contrast when imaging second phases in crystalline materials using transmission electron microscopy. With the suggested name of plasmon-ratio imaging, the technique uses an energy-filtered imaging system to record and determine a ratio for two images taken at energies in the low loss region. Unlike core-loss imaging, the use of very thin specimens is not required. It is concluded that it is often possible to create a ratio image in which the contrast is dominated by energy loss, that is, chemical differences, rather than by diffraction effects.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

We present and review dopant mapping examples in semiconductor device structures by electron holography and outline their potential applications for experimental investigation of two-dimensional (2D) dopant diffusion on the nanometer scale. We address the technical challenges of the method when applied to transistor structures with respect to quantification of the results in terms of the 2D p–n junction potential and critically review experimental boundary conditions, accuracy, and potential pitfalls. By obtaining maps of the inner electrostatic potential before and after anneals typically used in device processing, we demonstrate how the “vertical” and “lateral” redistribution of boron during device fabrication can directly be revealed. Such data can be compared with the results of process simulation to extract the fundamental parameters for dopant diffusion in complex device structures.

Nb–silicide in situ composites have great potential for high-temperature turbine applications. Nb–silicide composites consist of a ductile Nb-based solid solution together with high-strength silicides, such as Nb5Si3 and Nb3Si. With the appropriate addition of alloying elements, such as Ti, Hf, Cr, and Al, it is possible to achieve a promising balance of room-temperature fracture toughness, high-temperature creep performance, and oxidation resistance. In Nb–silicide composites generated from metal-rich binary Nb-Si alloys, Nb3Si is unstable and experiences eutectoid decomposition to Nb and Nb5Si3. At high Ti concentrations, Nb3Si is stabilized to room temperature, and the eutectoid decomposition is suppressed. However, the effect of both Ti and Hf additions in quaternary alloys has not been investigated previously. The present article describes the discovery of a low-temperature eutectoid phase transformation during which (Nb)3Si decomposes into (Nb) and (Nb)5Si3, where the (Nb)5Si3 possesses the hP16 crystal structure, as opposed to the tI32 crystal structure observed in binary Nb5Si3. The Ti and Hf concentrations were adjusted over the ranges of 21 to 33 (at.%) and 7.5 to 33 (at.%) to understand the effect of bulk composition on the phases present and the eutectoid phase transformation.

Results for the X-ray emission efficiency (counts per C per sr) of K-lines for selected elements (C, Al, Si, Ti, Cu, Ge) and for the first time also for compounds and alloys (SiC, GaP, AlCu, TiAlC) are presented. An energy dispersive X-ray spectrometer (EDS) of known detection efficiency (counts per photon) has been used to record the spectra at a takeoff angle of 25° determined by the geometry of the secondary electron microscope's specimen chamber. Overall uncertainty in measurement could be reduced to 5 to 10% in dependence on the line intensity and energy. Measured emission efficiencies have been compared with calculated efficiencies based on models applied in standardless analysis. The widespread XPP and PROZA models give somewhat too low emission efficiencies. The best agreement between measured and calculated efficiencies could be achieved by replacing in the modular PROZA96 model the original expression for the ionization cross section by the formula given by Casnati et al. (1982) A discrepancy remains for carbon, probably due to the high overvoltage ratio.

Electron microprobe testing and evaluation procedures to determine the extent of within- and between-specimen heterogeneity of reference materials and the experimental uncertainty are described. These procedures have been developed and used at NIST in the certification of several NIST Standard Reference Materials (SRMs). In this article, they have been simplified and updated for general use. Suggestions for experimental testing of specimens are described and a detailed description of the statistical evaluation process is included with an example of a data spreadsheet and instructions for its preparation.

Following is a list of microscopy-related meetings and courses. The editors would greatly appreciate input to this list via the electronic submission form found in the MSA World-Wide Web page at http://www.msa.microscopy.com. We will gladly add hypertext links to the notice on the web and insert a listing of the meeting in the next issue of the Journal. Send comments and questions to JoAn Hudson, [email protected] or Nestor Zaluzec, [email protected].