A one-day pre-meeting workshop was held on August 7, 2011, immediately preceding the annual Microscopy and Microanalysis (M&M) 2011 meeting in Nashville, Tennessee. The workshop was organized by the Aberration-Corrected Electron Microscopy (ACEM) Focused Interest Group, with an Organizing Committee consisting of Phil Batson, Dave Muller, Larry Allard, Paul Voyles, Miofang Chi, and Mike O'Keefe.

Aberration correction, after a 50 year incubation period of developing ideas and techniques while awaiting enabling technology, has transformed electron microscopy during the first dozen years of the 21st century. Some of the conditions that accompanied this transformation, the required complexity and its effect on the way microscopy is pursued, recent results that promise to change the field, and directions for the future are briefly described.

In prior research, specimen holders that employ a novel MEMS-based heating technology (AduroTM) provided by Protochips Inc. (Raleigh, NC, USA) have been shown to permit sub-Ångström imaging at elevated temperatures up to 1,000°C during in situ heating experiments in modern aberration-corrected electron microscopes. The Aduro heating devices permit precise control of temperature and have the unique feature of providing both heating and cooling rates of 106°C/s. In the present work, we describe the recent development of a new specimen holder that incorporates the Aduro heating device into a “closed-cell” configuration, designed to function within the narrow (2 mm) objective lens pole piece gap of an aberration-corrected JEOL 2200FS STEM/TEM, and capable of exposing specimens to gases at pressures up to 1 atm. We show the early results of tests of this specimen holder demonstrating imaging at elevated temperatures and at pressures up to a full atmosphere, while retaining the atomic resolution performance of the microscope in high-angle annular dark-field and bright-field imaging modes.

The high beam current and subangstrom resolution of aberration-corrected scanning transmission electron microscopes has enabled electron energy loss spectroscopy (EELS) mapping with atomic resolution. These spectral maps are often dose limited and spatially oversampled, leading to low counts/channel and are thus highly sensitive to errors in background estimation. However, by taking advantage of redundancy in the dataset map, one can improve background estimation and increase chemical sensitivity. We consider two such approaches—linear combination of power laws and local background averaging—that reduce background error and improve signal extraction. Principal component analysis (PCA) can also be used to analyze spectrum images, but the poor peak-to-background ratio in EELS can lead to serious artifacts if raw EELS data are PCA filtered. We identify common artifacts and discuss alternative approaches. These algorithms are implemented within the Cornell Spectrum Imager, an open source software package for spectroscopic analysis.

TEAM I is the final product of the Transmission Electron Aberration-corrected Microscope (TEAM) Project, a collaborative project funded by the Department of Energy with the goal of designing and building a platform for a next generation aberration-corrected electron microscope capable of image resolution of up to 50 pm. The TEAM instrument incorporates a number of new technologies, including spherical- and chromatic-aberration correction, an all-piezo-electric sample stage and an active-pixel direct electron detector. This article describes the functionality of this advanced instrumentation, its response to changes in environment or operating conditions, and its stability during daily operation within the National Center for Electron Microscopy user facility.

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.

A new aberration-corrected scanning transmission electron microscope equipped with an array of Si-drift energy-dispersive X-ray spectrometers has been utilized to acquire spectral image data at atomic resolution. The resulting noisy data were subjected to multivariate statistical analysis to noise filter, remove an unwanted and partially overlapping non-sample-specific X-ray signal, and extract the relevant correlated X-ray signals (e.g., channels with L and K lines). As an example, the Y2Ti2O7 pyrochlore-structured oxide (assumed here to be ideal) was interrogated at the [011] projection. In addition to pure columns of Y and Ti, at this projection, there are also mixed 50-50 at. % Y-Ti columns. An attempt at atomic-resolution quantification is presented. The method proposed here is to subtract the non-column-specific signal from the elemental components and then quantify the data based upon an internally derived k-factor. However, a theoretical basis to predict this non-column-specific signal is needed to make this generally applicable.

The resolution of conventional electron microscopes is usually limited by spherical aberration. Microscopes equipped with aberration correctors are then primarily limited by higher order, chromatic, and misalignment aberrations. In particular the Nion third-order aberration correctors installed on machines with a low energy spread and possessing sophisticated alignment software were limited by the uncorrected fifth-order aberrations. Here we show how the Nion fifth-order aberration corrector can be used to adjust and reduce some of the fourth- and fifth-order aberrations in a probe-corrected scanning transmission electron microscope.

A method for measurement of the aberration status from high-resolution dark-field images is developed using scanning transmission electron microscopy (STEM), called the Segmented Image Autocorrelation function Matrix (SIAM). The method employs an autocorrelation function from the segmented area in the defocused STEM images from an aligned crystalline specimen to measure the defocus and twofold astigmatism for the probe-forming system. The values measured using this method can be fed directly back to the instrument by changing the strength of the stigmator and the objective lens of the microscope. It is successfully demonstrated that the feedback system can automatically correct the defocus and twofold astigmatism of the microscope after several iterations using practical STEM images from an actual crystalline specimen.

Atomic-size vortex beams have great potential in probing the magnetic moment of materials at atomic scales. However, the limited depth of field of vortex beams constrains the probing depth in which the helical phase front is preserved. On the other hand, electron channeling in crystals can counteract beam divergence and extend the vortex beam without disrupting its topological charge. Specifically, in this article, we report that atomic vortex beams with topological charge ±1 can be coupled to the 2p columnar bound states and propagate for more than 50 nm without being dispersed and losing its helical phase front. We give numerical solutions to the 2p columnar orbitals and tabulate the characteristic size of the 2p states of two typical elements, Co and Dy, for various incident beam energies and various atomic densities. The tabulated numbers allow estimates of the optimal convergence angle for maximal coupling to 2p columnar orbital. We have also developed analytic formulae for beam energy, convergence angle, and hologram-dependent scaling for various characteristic sizes. These length scales are useful for the design of pitch-fork apertures and operations of microscopes in the vortex-beam imaging mode.

An accurate determination of specimen thickness is essential for quantitative analytical electron microscopy. Here we demonstrate that a position-averaged incoherent bright-field signal recorded on an absolute scale can be used to determine the thickness of on-axis crystals with a precision of ±1.6 nm. This method measures both the crystalline and the noncrystalline parts (surface amorphous layers) of the sample. However, it avoids the systematic error resulting from surface plasmon contributions to the inelastic mean-free-path thickness estimated by electron energy loss spectroscopy.

The ability to see, or visualize, a phenomenon is an essential tool of modern biological research. Our ability to create static and dynamic images has grown exponentially in the 25 or so years since confocal microscopy became readily available. The ingenious and energetic application of insights from optical physics to biological imaging in recent years has brought us far-reaching extensions of simple imaging, including nonlinear (or multiphoton) excitation, total internal reflection imaging, and even single molecule counting techniques. The annual Omaha Imaging Symposium has since 2003 brought together experts in advanced biological imaging techniques for a one-day exposition of how these techniques help move biological science forward. The seventh iteration of the series was held Friday, April 8, 2011, at the Harper Student Life Center of Creighton University, in Omaha, Nebraska. This special section of Microscopy and Micronanalysis consists of papers from speakers at the symposium.

The immune system is highly evolved and can respond to infection throughout the body. Pathogen-specific immune cells are usually generated in secondary lymphoid tissues (e.g., spleen, lymph nodes) and then migrate to sites of infection where their functionality is shaped by the local milieu. Because immune cells are so heavily influenced by the infected tissue in which they reside, it is important that their interactions and dynamics be studied in vivo. Two-photon microscopy is a powerful approach to study host-immune interactions in living tissues, and recent technical advances in the field have enabled researchers to capture movies of immune cells and infectious agents operating in real time. These studies have shed light on pathogen entry and spread through intact tissues as well as the mechanisms by which innate and adaptive immune cells participate in thwarting infections. This review focuses on how two-photon microscopy can be used to study tissue-specific immune responses in vivo, and how this approach has advanced our understanding of host-immune interactions following infection.

Advances in microscopy allow one to probe the structure of neurons and their interactions with astrocytes in brain slices and in vivo at ever increasing resolution. Moreover, the dynamic interactions between the cells can be examined in live preparation. In this paper we discuss how a variety of imaging approaches: confocal microscopy, electron microscopy, and multiphoton time-lapse microscopy are employed to probe neuron glia interactions in the developing cerebellum.

The synaptic ribbon is a unique presynaptic structure with an intricate morphology in photoreceptors. Because of the resolution limit in conventional fluorescence microscopy, investigating ribbon protein locations has been challenging, especially in the early development stages of model animals. Here, we used stimulated emission depletion microscopy, a super-resolution imaging technique, to look at retina sections in 4 days post-fertilization (dpf) zebrafish. We observed that in photoreceptor cells, RIBEYE and RIM2 are expressed along the synaptic ribbon, with RIM2 consistently located inside of the horseshoe-shaped synaptic ribbon structure with RIBEYE located on the outside. The L-type calcium channel subunit, CACNA1F, exhibited small spot-like staining beneath the RIM2 and RIBEYE structures. Using morpholino antisense oligonucleotides to knock down RIBEYE expression, we observed fewer and shorter ribbons in the photoreceptor outer plexiform layers of 4 dpf fish retina as well as a reduction in RIM2 expression. The clustering of CACNA1F in these blind fish was no longer observed, but instead showed a diffuse expression in the photoreceptor terminal.

Confocal fluorescence microscopy is a broadly used imaging technique that enhances the signal-to-noise ratio by removing out of focal plane fluorescence. Confocal microscopes come with a variety of modifications depending on the particular experimental goals. Microscopes, illumination pathways, and light collection were originally focused upon obtaining the highest resolution image possible, typically on fixed tissue. More recently, live-cell confocal imaging has gained importance. Since measured signals are often rapid or transient, thus requiring higher sampling rates, specializations are included to enhance spatial and temporal resolution while maintaining tissue viability. Thus, a balance between image quality, temporal resolution, and tissue viability is needed. A subtype of confocal imaging, termed swept field confocal (SFC) microscopy, can image live cells at high rates while maintaining confocality. SFC systems can use a pinhole array to obtain high spatial resolution, similar to spinning disc systems. In addition, SFC imaging can achieve faster rates by using a slit to sweep the light across the entire image plane, thus requiring a single scan to generate an image. Coupled to a high-speed charge-coupled device camera and a laser illumination source, images can be obtained at greater than 1,000 frames per second while maintaining confocality.

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.

Recent technical advances have enabled the imaging of single fluorescent molecules. The application of single molecule visualization techniques has opened up new avenues of experimentation in biology at the molecular level. In this article, we review the application of single fluorescent molecule visualization and analysis to an important problem, that of subunit stoichiometry in membrane proteins, with particular emphasis on our approach. Single fluorescent molecules, coupled to fluorescent proteins, are localized in the membranes of cells. The molecules are then exposed to continuous low-level excitation until their fluorescent emissions reach background levels. The high sensitivity of modern instrumentation has enabled direct observations of discrete step decreases in the fluorescence of single molecules, which represent the bleaching of single fluorophores. By counting the number of steps over a large number of single molecules, an average step count is determined from which the stoichiometry is deduced using a binomial model. We examined the stoichiometry of a protein, prestin, that is central to mammalian hearing. We discuss how we prepared, identified, and imaged single molecules of prestin. The methodological considerations behind our approach are described and compared to similar procedures in other laboratories.

Trajectories of individual molecules moving within complex environments such as cell cytoplasm and membranes or semiflexible polymer networks provide invaluable information on the organization and dynamics of these systems. However, when such trajectories are obtained from a sequence of microscopy images, they can be distorted due to the fact that the tracked molecule exhibits appreciable directed motion during the single-frame acquisition. We propose a new model of image formation for mobile molecules that takes the linear in-frame motion into account and develop an algorithm based on the maximum likelihood approach for retrieving the position and velocity of the molecules from single-frame data. The position and velocity information obtained from individual frames are further fed into a Kalman filter for interframe tracking of molecules that allows prediction of the trajectory of the molecule and further improves the precision of the position and velocity estimates. We evaluate the performance of our algorithm by calculations of the Cramer-Rao Lower Bound, simulations, and model experiments with a piezo-stage. We demonstrate tracking of molecules moving as fast as 7 pixels/frame (12.6 μm/s) within a mean error of 0.42 pixel (37.43 nm).

Although imperfect spatial localization in single-molecule object tracking experiments has long been recognized to induce apparent motion in an immobile population of molecules, this effect is often ignored or incorrectly analyzed for mobile molecules. In particular, apparent motion due to positional uncertainty is often incorrectly assigned as a distinct diffusive mode. Here we show that, due to both static and dynamic contributions, positional uncertainty does not introduce a new apparent diffusive mode into trajectories, but instead causes a systematic shift of each measured diffusion coefficient. This shift is relatively simple: a factor of σ2/Δt is added to each diffusion coefficient, where σ is the positional uncertainty length scale and Δt is the time interval between observations. Therefore, by calculating the apparent diffusion coefficients as a function of Δt, it is straightforward to separate the true diffusion coefficients from the effective positional uncertainty. As a concrete demonstration, we apply this approach to the diffusion of the protein fibrinogen adsorbed to a hydrophobic surface, a system that exhibits three distinct modes of diffusion.