A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (τHNSCC = 1.21 ± 0.04 ns) than the surrounding normal tissue (τN = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

We demonstrate a new sensitive biosensor for detection of vital fungal spores of Aspergillus niger. The biosensor is based on silicon microfabricated cantilever arrays operated in dynamic mode. The change in resonance frequency of the sensor is a function of mass binding to the cantilever surface. For specific A. niger spore immobilization on the cantilever, each cantilever was individually coated with anti-Aspergillus niger polyclonal antibodies. We demonstrate the detection of single A. niger spores and their subsequent growth on the functionalized cantilever surface by online measurements of resonance frequency shifts. The new biosensor operating in humid air allows quantitative and qualitative detection of A. niger spores as well as detection of vital, functional spores in situ within ∼4 h. The detection limit of the sensor is 103 CFU mL−1. Mass sensitivity of the cantilever sensor is ∼53 pg Hz−1.

We report variable resolution fluctuation electron microscopy (VRFEM) measurements on Cu64.5Zr35.5 metallic glass acquired using scanning transmission electron microscopy nanodiffraction using coherent probes 0.8 to 11 nm in diameter. The VRFEM results show that medium range atomic order structure of Cu64.5Zr35.5 bulk metallic glass at the ∼1 nm scale has large fluctuations, but the structure becomes almost completely homogeneous at the 11 nm scale. We show that our experimental VRFEM data are consistent with two different models, the pair persistent model and the amorphous/nanocrystal composite model. We also report a new way to filter VRFEM data to eliminate the effect of specimen thickness gradient using high-angle annular dark field images as references.

The use of analytical spectroscopies during scanning/transmission electron microscope (S/TEM) investigations of micro- and nano-scale structures has become a routine technique in the arsenal of tools available to today’s materials researchers. Essential to implementation and successful application of spectroscopy to characterization is the integration of numerous technologies, which include electron optics, specimen holders, and associated detectors. While this combination has been achieved in many instrument configurations, the integration of X-ray energy-dispersive spectroscopy and in situ liquid environmental cells in the S/TEM has to date been elusive. In this work we present the successful incorporation/modifications to a system that achieves this functionality for analytical electron microscopy.

We show that aberration-corrected scanning transmission electron microscopy operating at low accelerating voltages is able to analyze, simultaneously and with single atom resolution and sensitivity, the local atomic configuration, chemical identities, and optical response at point defect sites in monolayer graphene. Sequential fast-scan annular dark-field (ADF) imaging provides direct visualization of point defect diffusion within the graphene lattice, with all atoms clearly resolved and identified via quantitative image analysis. Summing multiple ADF frames of stationary defects produce images with minimized statistical noise and reduced distortions of atomic positions. Electron energy-loss spectrum imaging of single atoms allows the delocalization of inelastic scattering to be quantified, and full quantum mechanical calculations are able to describe the delocalization effect with good accuracy. These capabilities open new opportunities to probe the defect structure, defect dynamics, and local optical properties in 2D materials with single atom sensitivity.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

The optimum imaging of an object structure at the sub-angstrom length scale requires precise adjustment of the lens aberrations of a high-resolution instrument up to the fifth order. A least-squares optimization of defocus aberration C1, third-order spherical aberration C3, and fifth-order spherical aberration C5 yields two sets of aberration coefficients for strong phase contrast up to the information limit: one for variable C1 and C3, at fixed C5, another for variable C1, C3, and C5. An additional correction to the defocus aberration, dependent on object thickness, is described, which becomes important for the use of image simulation programs in predicting optimum high-resolution contrast from thin objects at the sub-angstrom scale. For instruments with a sub-angstrom information limit the ultimate structure resolution, the power to resolve adjacent atom columns in a crystalline object, depends on both the instrumental pointspread and an object pointspread due to finite width of the atomic column potentials. A simulation study on a simple double-column model yields a range for structure resolutions, dependent on the atomic scattering power, from 0.070 nm down to 0.059 nm, for a hypothetical 300-kV instrument with an information limit of 0.050 nm.

Heterogeneous catalysis is one of the oldest nanosciences. Although model catalysts can be designed, synthesized, and, to a certain degree, characterized, industrial heterogeneous catalysts are often chemically and physically complex systems that have been developed through many years of catalytic art, technology, and science. The preparation of commercial catalysts is generally not well controlled and is often based on accumulated experiences. Catalyst characterization is thus critical to developing new catalysts with better activity, selectivity, and/or stability. Advanced electron microscopy, among many characterization techniques, can provide useful information for the fundamental understanding of heterogeneous catalysis and for guiding the development of industrial catalysts. In this article, we discuss the recent developments in applying advanced electron microscopy techniques to characterizing model and industrial heterogeneous catalysts. The importance of understanding the catalyst nanostructure and the challenges and opportunities of advanced electron microscopy in developing nanostructured catalysts are also discussed.

We have used fluctuation microscopy to reveal the presence of structural order on length scales of 1–2 nm in metallic glasses. We compare results of fluctuation microscopy measurements with high resolution transmission electron microscopy and electron diffraction observations on a series of metallic glass samples with differing degrees of structural order. The agreement between the fluctuation microscopy results and those of the other techniques is good. In particular, we show that the technique used to make thin specimens for electron microscopy affects the structure of the metallic glass, with ion thinning inducing more structural order than electropolishing. We also show that relatively minor changes in the composition of the alloy can have a significant effect on the medium-range order; this increased order is correlated with changes in mechanical behavior.

During laser deposition of Ni-Cr-B-Si-C alloys with high amounts of Cr and B, various microstructures and phases can be generated from the same chemical composition that results in heterogeneous properties in the clad layer. In this study, the microstructure and phase constitution of a high-alloy Ni-Cr-B-Si-C coating deposited by laser cladding were analyzed by a combination of several microscopy characterization techniques including scanning electron microscopy in secondary and backscatter imaging modes, energy dispersive spectroscopy (EDS), electron backscatter diffraction (EBSD), and transmission electron microscopy (TEM). The combination of EDS and EBSD allowed unequivocal identification of micron-sized precipitates as polycrystalline orthorhombic CrB, single crystal tetragonal Cr5B3, and single crystal hexagonal Cr7C3. In addition, TEM characterization showed various equilibrium and metastable Ni-B, Ni-Si, and Ni-Si-B eutectic products in the alloy matrix. The findings of this study can be used to explain the phase formation reactions and to tune the microstructure of Ni-Cr-B-Si-C coatings to obtain the desired properties.

Abstract: The theoretical interpretation of lattice resolution in high-angle annular dark-field images produced in a scanning transmission electron microscope (STEM) has been a subject of controversy. A first-order perturbation theoretical analysis is presented here, which shows that the contrast in the image arises from large-angle multiphonon, incoherent scattering, which is atomic number dependent. The lattice resolution is a consequence of coherently filling the objective aperture, and dynamical elastic diffraction preceding the large-angle multiphonon scattering is not a necessary requirement. Elastic scattering to the higher order Laue zone (HOLZ) is also shown to be negligible, compared with the incoherent scattering. Calculations from application of the theory are also presented. They show that lattice images formed using the high-angle annular dark-field detector are sensitive to atomic number and are relatively insensitive to defocus. Although high-angle annular dark-field lattice imaging appears to be simple, scattering into the high-angle detector can only be approximately described by an incoherent imaging model.

This study compared dentinal tubule density and diameter of human primary and permanent teeth at different depths of the coronal dentin. Crowns of eight primary second molars and eight permanent third molars were serially sectioned into three disks of ~0.5 mm thickness (superficial, middle, and deep layers), perpendicular to the long axis. Tubule density and diameter were evaluated in 2,000× and 3,000× magnifications by scanning electron microscopy. Data obtained were subjected to two-way repeated measures ANOVA and Tukey's post hoc test (α = 0.05). Tubule density was greater in primary teeth compared with permanent ones, regardless of depth (primary: 124,329 ± 43,594 mm2; permanent: 45,972 ± 21,098 mm2). In general, the tubule density increased as the dentin depth increased, except to the superficial and middle layers from permanent teeth. Tubule diameter was larger in the dentin layer close to the pulp chamber (superficial: 2.4 ± 0.07 μm; middle: 3.70 ± 0.06 μm; deep: 4.28 ± 0.04 μm). No difference was observed between primary (3.48 ± 0.81 μm) and permanent teeth (3.47 ± 0.73 μm). The tubule diameter increases as the dentin depth increases for primary and permanent teeth; however, the tubule density is higher in primary teeth.

At present, mechanisms by which specific structural and mechanical properties of the three-dimensional extracellular matrix microenvironment influence cell behavior are not known. Lack of such knowledge precludes formulation of engineered scaffolds or tissue constructs that would deliver specific growth-inductive signals required for improved tissue restoration. This article describes a new mechanical loading–imaging technique that allows investigations of structural–mechanical properties of biomaterials as well as the structural–mechanical basis of cell–scaffold interactions at a microscopic level and in three dimensions. The technique is based upon the integration of a modified, miniature mechanical loading instrument with a confocal microscope. Confocal microscopy is conducted in a reflection and/or fluorescence mode for selective visualization of load-induced changes to the scaffold and any resident cells, while maintaining each specimen in a “live,” fully hydrated state. This innovative technique offers several advantages over current biomechanics methodologies, including simultaneous visualization of scaffold and/or cell microstructure in three dimensions during mechanical loading; quantification of macroscopic mechanical parameters including true stress and strain; and the ability to perform multiple analyses on the same specimen. This technique was used to determine the structural–mechanical properties of three very different biological materials: a reconstituted collagen matrix, a tissue-derived biomaterial, and a tissue construct representing cells and matrix.

Persistence of gunshot residue (GSR) simultaneously collected from hands, face and hair, and clothing of the shooting person was examined. Samples were collected from five shooters in nine time intervals after a single shoot with a Luger 9 mm pistol, in the range of 0–4 h and examined with scanning electron microscopy and energy dispersive X-ray spectrometry. Numbers of particles, frequencies of occurrence of certain compositions of particles, and their sizes in function of the time intervals were inspected. The greatest numbers of particles were observed in samples collected from hands right after shooting, but they decrease quickly with time. In samples collected from the face smaller initial numbers of particles were found, but they lasted at a similar level longer. The estimated half-life times of particles were less than 1 h for samples taken from the hands, over 1 h for clothing and about 2–3 h for the face. In samples collected at longer intervals after shooting, there were particles present of small sizes and irregular shapes. The results demonstrate that including evidence collected from the suspect's face and hair may increase the probability of detection of GSR in cases when the suspect has not been apprehended immediately after the investigated incident.

The influence of W on the temporal evolution of γ′ precipitation toward equilibrium in a model Ni-Al-Cr alloy is investigated by three-dimensional atom-probe (3DAP) microscopy and transmission electron microscopy (TEM). We report on the alloys Ni-10 Al-8.5 Cr (at.%) and Ni-10 Al-8.5 Cr-2 W (at.%), which were aged isothermally in the γ + γ′ two-phase field at 1073 K, for times ranging from 0.25 to 264 h. Spheroidal-shaped γ′ precipitates, 5–15 nm diameter, form during quenching from above the solvus temperature in both alloys at a high number density (∼1023 m−3). As γ′ precipitates grow with aging at 1073 K, a transition from spheriodal- to cuboidal-shaped precipitates is observed in both alloys. The elemental partitioning and spatially resolved concentration profiles across the γ′ precipitates are obtained as a function of aging time from three-dimensional atom-by-atom reconstructions. Proximity histogram concentration profiles (Hellman et al., 2000) of the quaternary alloy demonstrate that W concentration gradients exist in γ′ precipitates in the as-quenched and 0.25-h aging states, which disappear after 1 h of aging. The diffusion coefficient of W in γ′ is estimated to be 6.2 × 10−20 m2 s−1 at 1073 K. The W addition decreases the coarsening rate constant, and leads to stronger partitioning of Al to γ′ and Cr to γ.

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.

The successful correction of spherical aberration is an exciting and revolutionary development for the whole field of electron microscopy. Image interpretability can be extended out to sub-Ångstrom levels, thereby creating many novel opportunities for materials characterization. Correction of lens aberrations involves either direct (online) hardware attachments in fixed-beam or scanning TEM or indirect (off-line) software processing using either off-axis electron holography or focal-series reconstruction. This review traces some of the important steps along the path to realizing aberration correction, including early attempts with hardware correctors, the development of online microscope control, and methods for accurate measurement of aberrations. Recent developments and some initial applications of aberration-corrected electron microscopy using these different approaches are surveyed. Finally, future prospects and problems are briefly discussed.

Atom probe tomography (APT) represents a significant step toward atomic resolution microscopy, analytically imaging individual atoms with highly accurate, though imperfect, chemical identity and three-dimensional (3D) positional information. Here, a technique to retrieve crystallographic information from raw APT data and restore the lattice-specific atomic configuration of the original specimen is presented. This lattice rectification technique has been applied to a pure metal, W, and then to the analysis of a multicomponent Al alloy. Significantly, the atoms are located to their true lattice sites not by an averaging, but by triangulation of each particular atom detected in the 3D atom-by-atom reconstruction. Lattice rectification of raw APT reconstruction provides unprecedented detail as to the fundamental solute hierarchy of the solid solution. Atomic clustering has been recognized as important in affecting alloy behavior, such as for the Al-1.1Cu-1.7Mg (at. %) investigated here, which exhibits a remarkable rapid hardening reaction during the early stages of aging, linked to clustering of solutes. The technique has enabled lattice-site and species-specific radial distribution functions, nearest-neighbor analyses, and short-range order parameters, and we demonstrate a characterization of solute-clustering with unmatched sensitivity and precision.