The design and construction of a double-hexapole aberration corrector has made it possible to build the prototype of a spherical-aberration corrected transmission electron microscope dedicated to high-resolution imaging on the atomic scale. The corrected instrument, a Philips CM200 FEG ST, has an information limit of better than 0.13 nm, and the spherical aberration can be varied within wide limits, even to negative values. The aberration measurement and the corrector control provide instrument alignments stable enough for materials science investigations. Analysis of the contrast transfer with the possibility of tunable spherical aberration has revealed new imaging modes: high-resolution amplitude contrast, extension of the point resolution to the information limit, and enhanced image intensity modulation for negative phase contrast. In particular, through the combination of small negative spherical aberration and small overfocus, the latter mode provides the high-resolution imaging of weakly scattering atom columns, such as oxygen, in the vicinity of strongly scattering atom columns. This article reviews further lens aberration theory, the principle of aberration correction through multipole lenses, aspects for practical work, and materials science applications.

A novel approach for nanoscale imaging and characterization of the orientation dependence of electromechanical properties—vector piezoresponse force microscopy (Vector PFM)—is described. The relationship between local electromechanical response, polarization, piezoelectric constants, and crystallographic orientation is analyzed in detail. The image formation mechanism in vector PFM is discussed. Conditions for complete three-dimensional (3D) reconstruction of the electromechanical response vector and evaluation of the piezoelectric constants from PFM data are set forth. The developed approach can be applied to crystallographic orientation imaging in piezoelectric materials with a spatial resolution below 10 nm. Several approaches for data representation in 2D-PFM and 3D-PFM are presented. The potential of vector PFM for molecular orientation imaging in macroscopically disordered piezoelectric polymers and biological systems is discussed.

With a vertical resolution of 0.1 nm, atomic force microscopy (AFM) height measurements can be used to determine accurately the diameter of single-walled carbon nanotubes (SWNT) with the assumption that they have circular cross sections. The aim of this article is to draw attention to the need to optimize operating parameters in tapping mode for quantitative AFM height (diameter) analysis of SWNTs. Using silicon tip/cantilever assemblies with force constants ranging from 0.9 to 40 N m−1, we examined the effect of applied force on the apparent diameter of SWNT wrapped with a 29-residue amphiphilic α-helical peptide. A decrease in apparent height (SWNT diameter) with increasing applied force was observed for the higher force constant cantilevers. Cantilevers having force constants of 0.9 and 3 N m−1 demonstrated minimal vertical sample compression with increasing applied force. The effects of AFM image pixel density and scan speed on the measured height (diameter) of SWNTs were also assessed.

Degradation processes occurring at the surface and in the bulk of Cu-based amorphous alloys during cathodic hydrogen charging were used for promoting the catalytic activity of such alloys. These processes modifying the structure, composition, and morphology of the substrate proved to be useful methods for transforming Cu-Hf and inactive Cu-Ti amorphous alloy precursors into active and durable catalysts. Indeed, their catalytic activity for dehydrogenation of 2-propanol increased up to a conversion level of ∼60% at selectivities to acetone of about 99% for Cu-Ti and to conversion of ∼90% at selectivities of ∼95% for Cu-Hf. Previous attempts carried out by aging in air or hydrogen charging from the gas phase resulted in a maximum conversion level up to 15% for Cu-Hf and up to 3% for Cu-Ti. High resolution Auger spectroscopy allowed changes occurring during the activation process to be identified, namely, the formation of small Cu particles on the HfO2 surface and the formation of highly porous particles containing mostly Cu and some Ti and O (Cu-Ti-O) on a Cu-Ti substrate. Differences in the chemistry and structure of both catalysts are discussed, and the implications for catalytic function are considered. A probable configuration of active sites on the Cu-Ti-O/Ti-O-Cu catalyst for dehydrogenation of 2-propanol is proposed.

Detection of Förster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater Förster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.Note: M. Rizzo and K. Segawa performed this research at Vanderbilt University (same address as Piston). W. Zipfel performed research at Cornell University.

In a conventional transmission electron microscope system, the resolution is regarded as an absolute limitation, that is, 0.2 nm in theory and 0.6 nm in sections of biological materials. However, in an oversampled system, this limitation can be broken. In the present study, 60-nm-thick Epon sections from a mouse kidney were used. From these sections tight junctions located in the distal tubule were selected as test objects. Sets of up to 15 electron microscope images of the same target were recorded on negatives at ×10,000, ×13,000, and ×63,000, respectively. The recorded films were digitized using a light microscope equipped with a digital camera. In each set the images were expanded, aligned, and merged into a more highly resolved output image. Each output image revealed details in the tight junction, which were not visible at the original magnifications. Two different sizes of colloidal gold particles (10 nm and 1 nm) conjugated with an immunoglobin G (IgG) served as references. With this improvement of resolution, it becomes possible to inspect some barely visible biologic (virus) particles and structures, such as glycogen and free ribosomes in their native environment.

Corrosion-casted capillary systems of the kidney glomerulus were imaged with confocal microscopy because of the fluorescence properties of the casting plastic. Acquisition of a z-series through the glomerular capillaries provided three-dimensional data sets from which surface-rendered models were generated. These models could be rotated and viewed from any angle and also contained quantitative information allowing cast surface area and volume measurements to be calculated. The computer-generated models were also skeletonized to form a one-voxel-thick skeleton of the original model. The skeleton exhibited the three-dimensional topology and network of the capillary bed, and interior capillary relations could also be viewed. Quantitative information such as the total capillary length and number of capillary intersects was calculated from the skeletonized model. Extending this method to noncorroded kidney specimens revealed not only the casted vessels but also cellular features of the adjacent tissues surrounding the capillaries.

Nuclear factor–kappa B (NF-κB) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-κBs, which prevent entry into the nucleus. Following cellular stimulation, the I-κBs are rapidly degraded, activating NF-κB. The active form of NF-κB rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-κB is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-κB (p65 subunit, p65-NF-κB) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-κB protein in the nucleus determined biochemically. The appearance of p65-NF-κB in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-κB can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

Date Change: Microscopy and Microanalysis 2006 July 30–August 3, 2006 Chicago, Illinois, USA

Sponsor: Microscopy Society of America (MSA), Microbeam Analysis Society (MAS), International Metallographic Society (IMS)

URL: www.msa.microscopy.com

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, joanh@clemson.edu or Nestor Zaluzec, zaluzec@aaem.amc.anl.gov.