For the first few centuries of microscopy, spatial resolution was limited by the diffraction barrier. Recently, this barrier has been broken using several different methods. Optical methods that provide better resolution than the diffraction barrier are referred to as super-resolution. Although these techniques have significantly improved resolution in two dimensions (x and y) or in the axial dimension (z), it has not been possible to achieve substantial improvement in all three dimensions simultaneously. A study by Bo Huang, Wenqin Wang, Mark Bates, and Xiaowei Zhuang demonstrated a breakthrough by achieving a spatial resolution that is 10 times better than the diffraction limit in all three dimensions without using sample or optical-beam scanning.

There are several “molecular machines” that have been devised on a nanometer scale, made from proteins, DNA, and other molecules. A molecular machine is a system that generates physical forces at the atomic level, controlled by an external stimulus. Since all of the proposed circuits connect components linearly, they only communicate with one machine at a time. Now, Anirban Bandyopadhyay and Somobrata Acharya have devised an ingenious device that has the potential to communicate different instructions to many molecular machines simultaneously. They demonstrated that 2,3,5,6-tetramethyl-1–4-benzoquinone (duroquinone; DRQ) can be assembled as 17 identical molecules, one central molecule surrounded radially by 16 others. The central molecule can control the conformation of all the others when switched to one of four logic states by suitable pulses from an atomic sharp needle of a scanning tunneling microscope (STM).

It was demonstrated 18 years ago that atoms could be manipulated, one at a time, on a surface. Yet only recently has the force required to move an atom been determined. Markus Ternes, Christopher Lutz, Cyrus Hirjibehedin, Franz Giessibl, and Andreas Heinrich, in a technical tour de force, have engineered a microscope that incorporates features of the scanning tunneling microscope (STM) and atomic force microscope (AFM) to accurately quantitate the lateral and vertical forces needed to move a single atom on a surface.

It is relatively infrequent these days for a novel mineral to be discovered in the natural environment (in this context, this implies on the Earth). On the rare events of such a discovery, the new mineral is typically found in milligram quantities. In an article by Ronald Peterson, William Nelson, Bruce Madu, and Herbert Shervell, they describe the discovery of kilograms of a mineral that had only been synthesized previously, but never before detected in the natural environment. As if that were not impressive enough, they went looking for this mineral because they thought it had been observed on Mars!

It has been appreciated that many invertebrates have morphologic and/or behavioral traits that help them survive by camouflage for predator avoidance or being stealthy predators themselves. The morphologic features that are involved have been described in greater detail as the technology has evolved. Recently, Rebecca Duncan, Kellar Autumn, and Greta Binford have used dissecting microscopes and scanning electron microscopes (SEM) to reveal new details of how certain species of spiders hide themselves by covering themselves with sand, and how the sand particles tenaciously stick to the spiders’ bodies.

Fluorescence microscopy has revolutionized cell biology. Not only has it increased the spatial resolution with which certain processes are visualized, but it has allowed microscopists to monitor more than one event at a time. One of the remaining challenges is to see how proteins fold and assume their native conformations within living cells. Recently, Nathan Luedtke, Rachel Dexter, Daniel Fried, and Alanna Schepartz have demonstrated a technique to demonstrate these events in situ.

In the now classic marketing text, Crossing the Chasm, Geoffrey Moore describes the difficult transition every successful new technology must make as it evolves from laboratory curiosity to mainstream acceptance. Essentially the transition consists of a radical shift in focus, from exploring the possibilities that fill the minds of visionaries and innovators to satisfying the practical concerns of pragmatists and conservatives who are more interested in integration with existing technologies and return on investment. Initial development of the atom probe was motivated primarily by scientific curiosity and the desire to do novel research with a new microscopy technique. As the technology matured the focus of development efforts shifted to expanding the applications space and improving practical performance in areas such as sensitivity, fidelity, productivity, and ease of use.

A key to understanding the function of biological systems is the visualization of their natural state, ideally in a natural environment. At a molecular level, this is challenging. Traditional experimental techniques, like X-ray crystallography, can provide the atomic structure of proteins, but only by removing them from their native surroundings and forcing them into crystals. Over the past decade, microscopy techniques have emerged as alternatives to these traditional structure determination methods, with the advantage of visualizing molecules in a near-native state. Given the current focus of structural biology on interactions between proteins and better understanding of large protein complexes, cryo-electron microscopy (cryo-EM) has become a valuable tool.

Although considerable advances have been made in Energy Dispersive Detectors for microanalysis, low energy analysis under 1000eV is still relatively poor due to detector response and inefficient production of low energy x-rays. X-ray optics fabrication methods by O’Hara and measurements by McCarthy et. al. indicated that it should be possible to fabricate x-ray optics that could be used to significantly increase the low energy x-ray flux seen by an EDS detector without increasing the beam current. Such an optic would be useful to increase low energy counts without moving the detector closer, which would simply increase the high energy counts and dead time.

Ice plays an important role in many naturally occurring phenomena. For example, most rainfall in temperate climates is triggered by the nucleation of ice around μm-sized particles in atmospheric clouds. Another reason to be interested in ice is that thin supported ice films provide excellent model systems for studying the interaction of water with solid surfaces. Despite the importance of water-solid interactions for catalysis, corrosion, water purification and fuel cells, even the basics of this interaction are poorly understood. In fact, the best theoretical models often fail to predict even the simplest phenomena. A fundamental question, for example, is whether water/ice wets a given substrate, i.e., whether the substrate is covered up by a uniformly thick water/ice film. (In the case of non-wetting an ice film would break up into separate three-dimensional (3D) crystallites of varying height, exposing the substrate.)

The discovery of viable microbial life in ancient glacial ice has opened up a plethora of questions related to how life can be sustained in such extreme environments. Cryo-microscopic analysis of lab prepared ice samples indicates that microbes are located in the small veins of water that exist between individual ice crystals. Crucial to these studies is to obtain an understanding of the physicochemical habitat for life through physiological and microscopic analysis.

The rapid technical development of FIM (Focused Ion Beam) technology has spawned an increase in spatial resolution capability in scanning ion microscopy (SIM) technology. Furthermore, FIM has been used for preparation of thin specimens in transmission electron microscopy and micro-fabrication of electronic devices in the semiconductor industry. Recently, a scanning ion microscope with a helium field ion source has been developed. Thus, the contrast formation of emission electron images in scanning ion microscopy has been the object of study for analyzing images of materials specimens, similar to the theory behind scanning electron microscope (SEM) contrast formation. Furthermore, whether the electron emission yield γ induced by ion impact is periodic or non-periodic as a function of Z2 (the atomic number of the target) has not been well studied in the low energy region from several keV to the several tens of keV values used in SIM.

Transmission electron microscopy (TEM) has been proven to be one of the most convenient and straight forward methods for resolving sub-nanometer structures. The use of these instruments in life sciences, materials sciences and in the semiconductor industry has contributed greatly to many significant advances. However, typical TEMs are very large, expensive and relatively complicated to operate. Fortunately, recent advances in TEM technology and product design have created a new TEM instrument that is very well suited to everyday applications in hospitals, universities and laboratories of all types. Able to fit in a small room, this new TEM does not require the expensive, extensive building modifications usually associated with a TEM installation.

Between the Biological and Materials sciences resides the food industry, where both biological and materials methods and techniques are employed for both production and analysis. Often referred to as “Black Specs,” one of the biggest concerns for the food industry is contamination of the food, or its additives and ingredients, with foreign material (FM). Many of our customers seek assistance in solving their FM issues, which may require multiple instrumentation, several methods, and considerable open communication with the customer before a satisfactory result can be achieved. An additional level of difficulty is often present when we learn that the FM is so small we can barely detect it with the unaided eye. Particularly difficult is what might be referred to as “chemical contamination.” We present here in example (and in the upcoming Microscopy and Microanalysis meeting in Albuquerque this August), several of the methods employed in assisting our customers with FM issues.

Like no other microscopy technique, atom-probe tomography (APT) requires detailed data analysis algorithms specific to the knowledge desired, as the data are both complex due to their three-dimensional nature and can only be collected in a digital format. With recent increases in speed and field of view available in contemporary instruments like the Imago Scientific Instruments LEAP™ microscopes, these challenges and significant benefits are exacerbated. In practice, ‘data collection’ in APT, as understood in complementary techniques like scanning electron microscopy (SEM) or transmission electron microscopy (TEM), does not even begin until after the atom-probe experiment is over and the microscopist leaves the laboratory. The sample is prepared into the appropriate needle-shaped geometry, field evaporated atom by atom, and the ‘experiment’ part of the specimen analysis is over as soon as the ions are detected and stored in a digital file.

During the past several years, ASTM Subcommittee E04.14 on Quantitative Metallography has been developing an Image Analysis (I/A) methodology to determine the degree of circularity, roundness or sphericity of graphite nodules in ductile cast iron. In the as-polished condition, ideally the iron nodules should appear as dark circular objects in a light gray matrix, Figure 1. When performing Image Analysis measurements for these types of materials, complex gray image procedures should not be required to properly threshold the images to obtain an acceptable binary image on which to perform the measurements.

High-content screening (HCS) and other quantitative cellular imaging assay methods that use fluorescence microscopy require effective fluorescent labeling and identification of the target cell(s). Fluorescent probes that stain cells as the primary objects are used to identify and count individual cells, as well as to define the region(s) of each cell to which target-specific image analysis is applied. For this purpose, the primary object can be a major component of the cell, such as the nucleus or a large organelle, or the whole cell itself. When the whole cell is the primary object, a high-quality cell stain is needed to delineate the intact cell from bordering cells without also interfering in target-specific detection and analysis. Thermo Scientific Cellomics ® Whole Cell Stains (Thermo Fisher Scientific Inc., Rockford, Ill.), available in blue, green, orange and red, provides image staining and the ability to quantify the whole cell volume in HCS and fluorescence microscopy assays.

Specimen preparation techniques have evolved hand in hand with microscopy since the first microscopes. Since the introduction of the first Electron Microscope (EM) in the 1930’s, the basic problem with biological electron microscopy has been to preserve the structure of soft condensed, hydrated matter (e.g. tissues, cells, proteins, etc.) so that they can be viewed in the harsh environment of the electron microscope's high vacuum and ionizing radiation. For this, cells must be “fixed” with chemical cross-linkers, commonly glutaraldehyde, formaldehyde or some combination of both, stained with heavy metals (osmium tetroxide that provides contrast of biological components), dehydrated with an organic solvent, and infiltrated with a resin for eventual thin-sectioning. Only then can it be viewed with the EM. Such treatment with chemical fixatives and stains remains the standard approaches to arrest biological processes in cells or tissues, but at the cost of introducing clearly recognizable artifacts.

X-ray ultramicroscopy in the SEM is a relatively new application in the wider field of X-ray microscopy. This latter field includes synchrotron and cabinet-based systems that vary in their X-ray power, capability, sample size, spatial resolution, and convenience. One important capability of the SEM-hosted X-ray microscope is that the normal SEM imaging and analytical functions such as secondary and backscattered imaging and microanalysis by EDX or WDS are unimpeded. X-ray imaging then serves as a complement to the normal use of the SEM. The convenience of easy access in an SEM lab to an X-ray microscope with 3D tomographic capability makes this an important development.

In order to understand the focusing action of a TEM objective lens, a simple imaging system (figure 1) is best considered. This system consists of an objective lens and a single projector. In operation, the projector is adjusted as required within the total imaging system to achieve a magnification, M2, on the screen. This results in a focal length of F1, with the lens seeking to find an image in the position M1. If the objective lens does not place the image at M1 the result on the screen is an out of focus condition. The objective lens may have produced an image short of M1, overfocus, or beyond M1, underfocus.