In many cases in experimental science, the instrument interface becomes a limiting factor in the efficacy of carrying out unusual experiments or prevents the complete understanding of the acquired data. We have developed an advanced interface for scanning probe microscopy (SPM) that allows intuitive rendering of data sets and natural instrument control, all in real time. The interface, called the nanoManipulator, combines a high-performance graphics engine for real-time data rendering with a haptic interface that places the human operator directly into the feedback loop that controls surface manipulations. Using a hand-held stylus, the operator moves the stylus laterally, directing the movement of the SPM tip across the sample. The haptic interface enables the user to “feel” the surface by forcing the stylus to move up and down in response to the surface topography. In this way the user understands the immediate location of the tip on the sample and can quickly and precisely maneuver nanometer-scale objects. We have applied this interface to studies of the mechanical properties of nanotubes and to substrate-nanotube interactions. The mechanical properties of carbon nanotubes have been demonstrated to be extraordinary. They have an elastic modulus rivaling that of the stiffest material known, diamond, while maintaining a remarkable resistance to fracture. We have used atomic-force microscopy (AFM) to manipulate the nanotubes through a series of configuration that reveal buckling behavior and high-strain resilience. Nanotubes also serve as test objects for nanometer-scale contact mechanics. We have found that nanotubes will roll under certain conditions. This has been determined through changes in the images and through the acquisition of lateral force during manipulation. The lateral force data show periodic stick-slip behavior with a periodicity matching the perimeter of the nanotube.

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Abstract: We compare here multi-photon excitation microscopy and tandem scanning reflected light confocal microscopy for the microscopic observation of human skin in vivo. Multi-photon excitation is induced by a 80-MHz pulse train of femtosecond laser pulses at 780 nm wavelength. This nonlinear microscopic technique is inherently suitable for tissue fluorescence imaging because of its deeper penetration depth and lower specimen photodamage. This technique has noninvasively obtained tissue structural information in human epidermis and dermis. Alternatively, tandem scanning confocal light microscopy based on a white light source can provide video-rate image acquisition with high resolution and high contrast. Reflected light confocal methods have been used to obtain images from the skin surface to the epidermal–dermal junction. The relative merits of these two techniques can be identified by comparing three-dimensionally resolved images obtained from the forearm skin of the same volunteer.

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Bacteria flourish in nearly every environment on earth. Contributing to their ability to grow in many esoteric locations is their development into a biofilm structure. In an effort to more accurately model the growth environment of biofilms in nature, a Center for Disease Control and Prevention (CDC) biofilm reactor has been developed that mimics nature-like shear forces and renewable nutrient sources. To date, there has been no confirmation by scanning electron microscopy (SEM) that mature biofilms develop on a surface when grown using the CDC biofilm reactor. Three different SEM methods were used to collect images of Staphylococcus epidermidis ATCC 35984 that was to be grown using the CDC biofilm reactor. In addition, two different fixative techniques were used in each of the imaging methods. Results indicated that after 48 hours of growth in the reactor, S. epidermidis ATCC 35984 does produce a significant network of matrix components and 3D mushroom- or pillar-like structures with signs of water channel development. In conclusion, S. epidermidis ATCC 35984 grown using the CDC biofilm reactor does appear to display signs of mature biofilm development. These results could be important for studies wherein mature biofilms are needed for in vitro and/or in vivo applications.

Patterning of cells is critical to the formation and function of the normal organ, and it appears to be dependent upon internal and external signals. Additionally, the formation of most tissues requires the interaction of several cell types. Indeed, both extracellular matrix (ECM) components and cellular components are necessary for three-dimensional (3-D) tissue formation in vitro. Using 3-D cultures we demonstrate that ECM arranged in an aligned fashion is necessary for the rod-shaped phenotype of the myocyte, and once this pattern is established, the myocytes were responsible for the alignment of any subsequent cell layers. This is analogous to the in vivo pattern that is observed, where there appears to be minimal ECM signaling, rather formation of multicellular patterns is dependent upon cell–cell interactions. Our 3-D culture of myocytes and fibroblasts is significant in that it models in vivo organization of cardiac tissue and can be used to investigate interactions between fibroblasts and myocytes. Furthermore, we used rotational cultures to examine cellular interactions. Using these systems, we demonstrate that specific connexins and cadherins are critical for cell–cell interactions. The data presented here document the feasibility of using these systems to investigate cellular interactions during normal growth and injury.

The morphology of the outer and inner membranes of traumatic chronic subdural hematomas (CSDHs) surgically removed from eight patients was investigated by scanning electron microscopy (SEM). Hematomas were divided into three groups based on time that had passed from the initiation of trauma to surgery. Structure of the CSDHs showed gradual morphological changes of the developing hematoma capsule. They initially included angiogenic and aseptic inflammatory reactions followed by progressive involvement of fibroblasts—proliferating and producing collagen fibrils. Numerous capillaries suggesting formation of new blood vessels were observed mainly in young hematomas removed between 15 and 21 days after trauma. In “older” hematomas (40 days after trauma), more numerous capillaries and thin-walled sinusoids were accompanied by patent, larger diameter blood vessels. Within the fibrotic outer membrane of the “oldest” hematoma capsules (60 or more days after trauma), especially in the area over the hematoma cavity, blood vessels were frequently occluded by clots. The results suggest dynamic changes in cellular and vascular organization of traumatic CSDH capsules paralleling the progression in hematoma age.

Atomic-scale tomography (AST) is defined and its place in microscopy is considered. Arguments are made that AST, as defined, would be the ultimate microscopy. The available pathways for achieving AST are examined and we conclude that atom probe tomography (APT) may be a viable basis for AST on its own and that APT in conjunction with transmission electron microscopy is a likely path as well. Some possible configurations of instrumentation for achieving AST are described. The concept of metaimages is introduced where data from multiple techniques are melded to create synergies in a multidimensional data structure. When coupled with integrated computational materials engineering, structure–properties microscopy is envisioned. The implications of AST for science and technology are explored.

High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Ångstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.

The ability to correctly track objects in time-lapse sequences is important in many applications of microscopy. Individual object motions typically display a level of dynamic regularity reflecting the existence of an underlying physics or biology. Best results are obtained when this local information is exploited. Additionally, if the particle number is known to be approximately constant, a large number of tracking scenarios may be rejected on the basis that they are not compatible with a known maximum particle velocity. This represents information of a global nature, which should ideally be exploited too. Some time ago, we devised an efficient algorithm that exploited both types of information. The tracking task was reduced to a max-flow min-cost problem instance through a novel graph structure that comprised vertices representing objects from three consecutive image frames. The algorithm is explained here for the first time. A user-friendly implementation is provided, and the specific relaxation mechanism responsible for the method's effectiveness is uncovered. The software is particularly competitive for complex dynamics such as dense antiparallel flows, or in situations where object displacements are considerable. As an application, we characterize a remarkable vortex structure formed by bacteria engaged in interstitial motility.

The Monte Carlo program PENEPMA performs simulations of X-ray emission from samples bombarded with both electron and photon beams. It is based on the general-purpose Monte Carlo simulation package PENELOPE, an elaborate system for the simulation of coupled electron-photon transport in arbitrary materials, and on the geometry subroutine package PENGEOM, which tracks particles through complex material structures defined by quadric surfaces. After a brief description of the interaction models implemented in the simulation subroutines and of the structure and operation of PENEPMA, we provide an overview of the capabilities of the program along with several examples of its application to the modeling of electron probe microanalysis measurements.

The damage layers generated in III–V compounds exposed to energetic gallium ions in a focused ion beam (FIB) instrument have been characterized by transmission electron microscopy (TEM). The damage on the side walls of the milled trenches is in the form of amorphous layers associated with direct amorphization from the gallium beam, rather than from redeposition of milled material. However, the damage on the bottom of the milled trenches is more complex. For InP and InAs the damage layers include the presence of crystalline phases resulting from recrystallization associated heating from the incident beam and gallium implantation. In contrast, such crystalline phases are not present in GaAs. The thicknesses of the damage layers are greater than those calculated from theoretical models of ion implantation. These differences arise because the dynamic nature of FIB milling means that the energetic ion beams pass through already damaged layers. In InP recoil phosphorus atoms also cause significant damage.

In contrast to symmetric division in mitosis, mammalian oocyte maturation is characterized by asymmetric cell division that produces a large egg and a small polar body. The asymmetry results from oocyte polarization, which includes spindle positioning, migration, and cortical reorganization, and this process is critical for fertilization and the retention of maternal components for early embryo development. Although actin dynamics are involved in this process, the molecular mechanism underlying this remained unclear until the use of confocal microscopy and live cell imaging became widespread in recent years. Information obtained through a PubMed database search of all articles published in English between 2000 and 2012 that included the phrases “oocyte, actin, spindle migration,” “oocyte, actin, polar body,” or “oocyte, actin, asymmetric division” was reviewed. The actin nucleation factor actin-related protein 2/3 complex and its nucleation-promoting factors, formins and Spire, and regulators such as small GTPases, partitioning-defective/protein kinase C, Fyn, microRNAs, cis-Golgi apparatus components, myosin/myosin light-chain kinase, spindle stability regulators, and spindle assembly checkpoint regulators, play critical roles in asymmetric cell division in oocytes. This review summarizes recent findings on these actin-related regulators in mammalian oocyte asymmetric division and outlines a complete signaling pathway.

The 50th anniversary of the application of electron probe microanalysis (EPMA) to the Earth Sciences provides an opportunity for an assessment of the state-of-the-art of the technique. Stemming from the introduction of the first automated instruments, the latest developments of EPMA and some typical applications are reviewed with an eye to the future. The most noticeable recent technical achievements such as the field-emission electron gun, the latest generation of energy and wavelength dispersive spectrometers, and the development of analytical methods based on new sets of first principle data obtained by the use of sophisticated computer codes, allow for the extension of the method to the analysis of trace elements, ultra-light elements (down to Li), small particles, and thin films, with a high degree of accuracy and precision and within a considerably reduced volume of interaction. A number of working examples and a thorough list of references provide the reader with a working knowledge of the capabilities and limitations of EPMA today.

Although the most familiar consequences of specimen charging in transmission electron microscopy can be eliminated by evaporating a thin conducting film (such as a carbon film) onto an insulating specimen or by preparing samples directly on such a conducting film to begin with, a more subtle charging effect still remains. We argue here that specimen charging is in this case likely to produce a dipole sheet rather than a layer of positive charge at the surface of the specimen. A simple model of the factors that control the kinetics of specimen charging, and its neutralization, is discussed as a guide for experiments that attempt to minimize the amount of specimen charging. Believable estimates of the electrostatic forces and the electron optical disturbances that are likely to occur suggest that specimen bending and warping may have the biggest impact on degrading the image quality at high resolution. Electron optical effects are likely to be negligible except in the case of a specimen that is tilted to high angle. A model is proposed to explain how both the mechanical and electron-optical effects of forming a dipole layer would have much greater impact on the image resolution in a direction perpendicular to the tilt axis, a well-known effect in electron microscopy of two-dimensional crystals.

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Electron energy loss spectra in conjunction with near-edge fine structures of purely stoichiometric niobium monoxide (NbO) and niobium pentoxide (Nb2O5) reference materials were recorded. The structures of the niobium oxide reference materials were checked by selected area electron diffraction to ensure a proper assignment of the fine structures. NbO and Nb2O5 show clearly different energy loss near-edge fine structures of the Nb-M4,5 and -M2,3 edges and of the O-K edge, reflecting the specific local environments of the ionized atoms. To distinguish the two oxides in a quantitative manner, the intensities under the Nb-M4,5 as well as Nb-M2,3 edges and the O-K edge were measured and their ratios calculated. k-factors were also derived from these measurements.

The increasing use of environmental transmission electron microscopy (ETEM) in materials science provides exciting new possibilities for investigating chemical reactions and understanding both the interaction of fast electrons with gas molecules and the effect of the presence of gas on high-resolution imaging. A gaseous atmosphere in the pole-piece gap of the objective lens of the microscope alters both the incoming electron wave prior to interaction with the sample and the outgoing wave below the sample. Whereas conventional TEM samples are usually thin (below 100 nm), the gas in the environmental cell fills the entire gap between the pole pieces and is thus not spatially localized. By using an FEI Titan environmental transmission electron microscope equipped with a monochromator and an aberration corrector on the objective lens, we have investigated the effects on imaging and spectroscopy caused by the presence of the gas.

High-resolution characterizations of intergranular attack in alloy 600 (Ni-17Cr-9Fe) exposed to 325°C simulated pressurized water reactor primary water have been conducted using a combination of scanning electron microscopy, NanoSIMS, analytical transmission electron microscopy, and atom probe tomography. The intergranular attack exhibited a two-stage microstructure that consisted of continuous corrosion/oxidation to a depth of ~200 nm from the surface followed by discrete Cr-rich sulfides to a further depth of ~500 nm. The continuous oxidation region contained primarily nanocrystalline MO-structure oxide particles and ended at Ni-rich, Cr-depleted grain boundaries with spaced CrS precipitates. Three-dimensional characterization of the sulfidized region using site-specific atom probe tomography revealed extraordinary grain boundary composition changes, including total depletion of Cr across a several nm wide dealloyed zone as a result of grain boundary migration.