The year 2009 marked the centenary of the birth of Otto Scherzer, one of the early pioneers of electron microscopy. Scherzer was the originator of the famous microscopy theorem that the spherical and chromatic aberrations of rotationally symmetric electron lenses were unavoidable. In honor of this centennial occasion, we organized a special memorial symposium during Microscopy & Microanalysis 2009, which was held in Richmond, Virginia, in late July. The introductory talks of the symposium presented a fascinating mix of first-hand accounts about working with Scherzer in Darmstadt and descriptions of the correction concepts and the early corrector prototypes that emerged from his group. Placed in this historical context, the latest advances in aberration correction for scanning and fixed-beam instruments that were presented in this symposium were all the more impressive and conveyed a vivid sense of history in the making. Applications of aberration correction to a broad range of materials were also highlighted in platform and poster presentations. This special issue of Microscopy and Microanalysis contains refereed contributions from the work presented at the symposium and thus provides a representative overview of the recent emergence of aberration-corrected electron microscopy (ACEM) and some of the prospects and challenges for this burgeoning field.

Otto Scherzer was one of the pioneers of theoretical electron optics. He was coauthor of the first comprehensive book on electron optics and was the first to understand that round electron lenses could not be combined to correct aberrations, as is the case in light optics. He subsequently was the first to describe several alternative means to correct spherical and chromatic aberration of electron lenses. These ideas were put into practice by his laboratory and students at Darmstadt and their successors, leading to the fully corrected electron microscopes now in operation.

The recent advent of a novel design of in situ heating technology for electron microscopes has permitted unprecedented control of elevated temperature studies of catalytic materials, particularly when coupled with the sub-Ångström imaging performance of a modern aberration-corrected scanning transmission electron microscope (STEM). Using micro-electro-mechanical-systems (MEMS)-based Aduro™ heating chips from Protochips, Inc. (Raleigh, NC, USA) allows nearly instantaneous heating and cooling of catalyst powders, avoiding effects of temperature ramping as experienced with standard heating stages. The heating technology also provides stable operation limited only by the inherent drift in the microscope stage, thus allowing full image resolution to be achieved even at elevated temperatures. The present study details the use of both the high X-Y spatial resolution in both dark-field and simultaneous bright-field imaging, along with the high resolution in Z (depth sectioning) provided by the large probe incidence semiangle in the aberration-corrected instrument, to characterize the evolution of microstructure in a commercial Au/Fe2O3 water-gas shift catalyst during elevated temperature treatment. The phenomenon of Au diffusion to the surface of hematite support particles to form discrete crystalline Au nanoparticles in the 1–2 nm size range, after a prior leaching treatment to remove surface Au species has been characterized.

Lowering the electron energy in the transmission electron microscope allows for a significant improvement in contrast of light elements and reduces knock-on damage for most materials. If low-voltage electron microscopes are defined as those with accelerating voltages below 100 kV, the introduction of aberration correctors and monochromators to the electron microscope column enables Ångstrom-level resolution, which was previously reserved for higher voltage instruments. Decreasing electron energy has three important advantages: (1) knock-on damage is lower, which is critically important for sensitive materials such as graphene and carbon nanotubes; (2) cross sections for electron-energy-loss spectroscopy increase, improving signal-to-noise for chemical analysis; (3) elastic scattering cross sections increase, improving contrast in high-resolution, zero-loss images. The results presented indicate that decreasing the acceleration voltage from 200 kV to 80 kV in a monochromated, aberration-corrected microscope enhances the contrast while retaining sub-Ångstrom resolution. These improvements in low-voltage performance are expected to produce many new results and enable a wealth of new experiments in materials science.

For the transmission electron aberration-corrected microscope (TEAM) initiative of five U.S. Department of Energy laboratories in the United States, a correction system for the simultaneous compensation of the primary axial aberrations, the spherical aberration Cs, and the chromatic aberration Cc has been developed and successfully installed. The performance of the resulting Cc /Cs-corrected TEAM instrument has been investigated thoroughly. A significant improvement of the linear contrast transfer can be demonstrated. The information about the instrument one obtains using Young's fringe method is compared for uncorrected, Cs-corrected, and Cc /Cs-corrected instruments. The experimental results agree well with simulations. The conclusions might be useful to others in understanding the process of image formation in a Cc /Cs-corrected transmission electron microscope.

Tilted illumination exit-wave restoration is compared for two aberration-corrected instruments at different accelerating voltages. The experimental progress of this technique is also reviewed and the significance of off-axial aberrations examined. Finally, the importance of higher order aberration compensation combined with careful correction of the lower order aberrations is highlighted.

High-angle annular dark-field and annular bright-field imaging experiments were carried out on an aberration-corrected transmission electron microscope. These techniques have been demonstrated on thin films of complex oxides Ba3.25La0.75Ti3O12 and on LaB6. The results show good agreement between theory and experiments, and for the case of LaB6 they demonstrate the detection of contrast from the B atoms in the annular bright-field images. Elemental mapping with electron-energy-loss spectroscopy has been used to deduce the distribution of Cr and Fe in a thin film of the complex oxide Bi2(Fe1/2Cr3/2)O6 at the unit cell level and the changes in the near-edge structure within the inequivalent regions in the crystalline unit cell. Energy-filtered images in the low-loss region of the energy-loss spectrum show contrast and resolution consistent with the modulation of the signals from elastic scattering. High-resolution contrast, mediated by phonon scattering, is observed for interband transitions. The limitations in terms of detection and signal are discussed.

The aberration-corrected scanning transmission electron microscope can provide information on nanostructures with sub-Ångström image resolution. The relatively intuitive interpretation of high-angle annular dark-field (HAADF) imaging technique makes it a popular tool to image a variety of samples and finds broad applications to characterizing nanostructures, especially when combined with electron energy-loss spectroscopy and X-ray energy-dispersive spectroscopy techniques. To quantitatively interpret HAADF images, however, requires full understanding of the various types of signals that contribute to the HAADF image contrast. We have observed significant intensity enhancement in HAADF images, and large expansion of lattice spacings, of surface atoms of atomically flat ZnO surfaces. The surface-resonance channeling effect, one of the electron-beam channeling phenomena in crystalline nanostructures, was invoked to explain the observed image intensity enhancement. A better understanding of the effect of electron beam channeling along surfaces or interfaces on HAADF image contrast may have implications for quantifying HAADF images and may provide new routes to utilize the channeling phenomenon to study surface structures with sub-Ångström spatial resolution.

Electron holography has been shown to allow a posteriori aberration correction. Therefore, an aberration corrector in the transmission electron microscope does not seem to be needed with electron holography to achieve atomic lateral resolution. However, to reach a signal resolution sufficient for detecting single light atoms and very small interatomic fields, the aberration corrector has turned out to be very helpful. The basic reason is the optimized use of the limited number of “coherent” electrons that are provided by the electron source, as described by the brightness. Finally, quantitative interpretation of atomic structures benefits from the holographic facilities of fine-tuning of the aberration coefficients a posteriori and from evaluating both amplitude and phase.

A lens system is proposed that not only provides spherical correction of the objective lens by charges that are induced on a thin foil, in the way proposed in a paper by Otto Scherzer [Optik56(2), 133–147, 1980], but also provides Zernike phase contrast by means of an appropriate phase shift of the scattered electrons within the foil. This system has the potential to provide strong phase contrast from very low spatial frequencies to frequencies above 1/(100 pm).

This article focuses on the development of a transparent and uniform understanding of possibilities for three-dimensional (3D) imaging in scanning transmission and confocal electron microscopes (STEMs and SCEMs), with an emphasis on the annular dark-field STEM (ADF-STEM), bright-field SCEM (BF-SCEM), and ADF-SCEM configurations. The incoherent imaging approximation and a 3D linear imaging model for ADF-STEM are reviewed. A 3D phase contrast model for coherent-SCEM as well as a pictorial way to find boundaries of information transfer in reciprocal space are reviewed and applied to both BF- and ADF-SCEM to study their 3D point spread functions and contrast transfer functions (CTFs). ADF-STEM is capable of detecting the depths of dopant atoms in amorphous materials but can fail for crystalline materials when channeling substantially modifies the electron propagation. For the imaging of extended (i.e., nonpointlike) features, ADF-STEM and BF-SCEM exhibit strong elongation artifacts due to the missing cone of information. ADF-SCEM shows an improvement over ADF-STEM/BF-SCEM due to its differential phase contrast eliminating slowly varying backgrounds, an effect that partially suppresses the elongation artifacts. However, the 3D CTF still has a cone of missing information that will result in some residual feature elongation as has been observed in A. Hashimoto et al., J Appl Phys160(8), 086101 (2009).

Lipid-containing nanostructures, in the form of solid lipid nanoparticles or iron oxide nanoparticles (NPs) coated with a lipid shell, were used as case studies for assessing and optimizing staining for transmission electron microscopy structural and compositional characterization. These systems are of paramount importance as drug delivery systems or as bio-compatible contrast agents. In particular, we have treated the systems with a negative (phospshotungstic acid) or with a positive (osmium tetroxide) staining agent. For iron-oxide NPs coated with the lipid shell, negative staining was more efficient with respect to the positive one. Nevertheless, in particular cases the combination of the two staining procedures provided more complete morphological and compositional characterization of the particles.

The three-dimensional (3D) keratin filament network of pancreatic carcinoma cells was investigated with different electron microscopical approaches. Semithin sections of high-pressure frozen and freeze substituted cells were analyzed with scanning transmission electron microscope (STEM) tomography. Preservation of subcellular structures was excellent, and keratin filaments could be observed; however, it was impossible to three-dimensionally track the individual filaments. To obtain a better signal-to-noise ratio in transmission mode, we observed ultrathin sections of high-pressure frozen and freeze substituted samples with low-voltage (30 kV) STEM. Contrast was improved compared to 300 kV, and individual filaments could be observed. The filament network of samples prepared by detergent extraction was imaged by high-resolution scanning electron microscopy (SEM) with very good signal-to-noise ratio using the secondary electron signal and the 3D structure could be elucidated by SEM tomography. In freeze-dried samples it was possible to discern between keratin filaments and actin filaments because the helical arrangement of actin subunits in the F-actin could be resolved. When comparing the network structures of the differently prepared samples, we found no obvious differences in filament length and branching, indicating that the intermediate filament network is less susceptible to preparation artifacts than the actin network.

The current prognostic parameters, including tumor volume, biochemistry, or immunohistochemistry, are not sufficient to reflect the properties of cancer cells that distinguish them from normal cells. Our focus is to evaluate the effects of a combination of microtubule-polymerizing Taxol® and -depolymerizing colchicine on IAR20 PC1 liver cells by measuring the surface fractal dimension as a descriptor of two-dimensional vascular geometrical complexity. The fractal dimension offers a rapid means of assessing cell shape. Furthermore, we show correlations of fractal dimensions of cell contours with the latent factors from our previously employed cell shape analysis.

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.

Multispectral analysis combined with the Periodic Acid-Schiff method was used to investigate cytological features of insoluble polysaccharides and changes in total insoluble polysaccharide content (TPC) during pollen development in rice, including four cytoplasmic male sterility lines (MSLs) and their corresponding fertility-maintaining lines (FMLs). The multispectral curves of the relative transmittance value (RTV) and the images of developing pollen cells were obtained across a range of successive wavelengths (400–720 nm). A minimum RTV was found near 550 nm indicating an absorption peak of the TPC. Thus, the TPC was measured using the RTV of 550 nm. In the four FMLs, the minimum TPC of developing pollen cells occurred at the late microspore stage, while the maximum TPC occurred at the mature pollen grain stage. The TPC levels of pollen cells were significantly higher in the four FMLs than in their corresponding MSLs during and after pollen abortion. Notably, a steep decrease of multispectral curves at 420 nm appeared before the occurrence of abortion, implying a marker associated with pollen abortion in rice. Our results will be helpful for exploring the changes in TPC during pollen ontogenesis in rice and provide a novel method for the study of bio-macromolecules.

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 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 Nan Yao, [email protected].