There is a solid consensus that new methods of structure fabrication, placement, and organization within the sub-10-nm resolution gap are urgently required to meet existing challenges in condensed matter, semiconductors, and biotechnologies. Standard top-down methods such as resist-based lithographies even used at the shortest available wavelengths have clearly identified limitations. On the other hand, bottom-up approaches, like scanning probe manipulation techniques, remain challenging when trying to generate reproducible, functional, and addressable nano-structures. Therefore, at the laboratory level new routes must be explored.

Many infectious diseases prevalent in the developing world, including malaria and tuberculosis, are difficult to diagnose on the basis of symptoms alone but can be accurately detected using microscope examination. Currently the expense, size, and fragility of optical microscopes impede their widespread use in resource-limited settings. Addressing these obstacles facing microscopy in the developing world is a pressing need; over 800,000 people, primarily children in Africa, die annually of malaria, and more than 1,500,000 people die annually of tuberculosis [1][2]. The aim of this study is to design and validate a microscope for use in the developing world that combines high-resolution imaging, extreme affordability, and long-term durability.

When living cells are far below the surface, they pose particularly complex problems for researchers trying to view dynamic life activities in the laboratory. Many fluorescence imaging systems rely on short-wavelength ultraviolet (UV) or blue light, which is then absorbed by the specimen and emitted as visible light. But living tissue scatters so much short-wavelength light that some of the emitted fluorescence from the region of interest does not reach the detector. The deeper the area of interest, the more severe this problem becomes. Indeed, for every specimen, there is a point at which so much scatter occurs that traditional fluorescence imaging techniques are no longer effective. Raising the intensity of the excitation light in order to get more light out of the system can itself be lethal for living systems, as it causes increased photobleaching and phototoxicity. Adding to the problem is the fact that in deep imaging, regions of the specimen above and below the focal plane that are not of interest are exposed to light, causing unwanted fluorescence. Finally, excessive scattering of the excitation light when imaging deep below the specimen's surface results in an image with poor signal-to-noise ratio. These images tend to look soft and dull instead of crisp and full of contrast. Assuming that the scientist's research protocol will not allow the use of thinner-cut sections, deep imaging is still possible via multiphoton microscopy.

Nanopatterning refers to the fabrication of nanometer-scale structures, meaning patterns with at least one lateral dimension between the size of an individual atom and approximately 100 nm. Direct Write or Maskless Lithography as discussed in this article refers to the use of a focused beam, either an ion beam or an electron beam, to create a patterned image directly into (etch), or on top of (deposition), the target material. Both electron beams and ion beams can be used together with gas injection technology to deposit three dimensional structures on the nanometer scale through the process of either electron beam assisted or ion beam assisted chemical vapor deposition (CVD). The deposition occurs only in the vicinity where the electron beam or ion beam is being scanned. Therefore, the deposit will follow the form of the scanned beam in two dimensions. This approach can be applied to produce three-dimensional objects by successively layering upon the two-dimensional pattern. In the case of ion beams in particular, the direct write process can also produce an etch pattern on the nanometer scale as the ion beam physically mills away the material via ion bombardment. This process can also be chemically enhanced for certain materials such as simultaneous use of water to selectively etch carbon.

The nucleolus represents a highly dynamic nuclear domain arising from an equilibrium between the level of ribosomal RNA synthesis and the efficiency of ribosomal RNA processing [1, 2]. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now demonstrate that it has additional functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response, and biogenesis of multiple ribonucleoprotein particles. Ribosome biogenesis is regulated throughout interphase and ceases during mitosis (Figure 1). Thus, there is a direct relationship between cell growth and nucleolar activities. Nucleoli are well known to be dramatically modified in cancer cells. Additionally, a large number of key proteins from both DNA- and RNA-containing viruses are localized in the nucleolus, including the human immunodeficiency virus (HIV)-1 Rev and Tat proteins. Targeting of viral proteins to the nucleolus not only facilitates virus replication, but may also be required for pathogenic processes. The nucleolus can also be considered a sensor of stress due to the redistribution of the ribosomal proteins in the nucleoplasm through its disruption.

Over the past decade, Atomic Force/Scanning Probe Microscopy (AFM/SPM) has emerged as the leading tool for investigations at the nanoscale – doing everything from imaging, to compositional differentiation, to explorations of molecular forces. However, aside from some interesting tweaks, add-ons and repackaging, the field has seen no fundamentally new instruments for several years. For the extremely high-resolution AFM/SPMs, there has literally been no completely new microscope for well over a decade. We report here on the new CypherTM AFM from Asylum Research (Figure 1). that delivers upgrades from the existing field of older AFM/SPMs on a variety of levels.

Scanning electron microscopy and X-ray analysis are standard tools in the nanotechnology world, but access to them by students is limited by high cost and lack of equipment. For this reason, among others, we have developed an on-demand remote access system to The University Spectroscopy and Imaging Facilities (USIF) of The University of Arizona (UA), by using Quartz PCI-Taipan. In general, Taipan is an online server based program that enables spectroscopy laboratories to store, organize, and manage data as well as users. Significantly, the Taipan system allows users, operators and researches to create their own online collaborations, which will, in many ways, fuse their research and outreach efforts. Therefore, we have incorporated the Taipan software to our recently installed Hitachi S-4800 FESEM and S-3400N variable-pressure SEM. With a high-speed internet connection, from any remote setting, the system allows students real-time access to view state-of-the-art microscopes, acquire data, and interact with the instrument operator. What is more, remote users can view up to six different screens including SEM image, SEM control screen, EDS, Raman spectrum, chamber scope, and microscope room as shown in Fig. 1. This offers a significant reduction of time, travel expenses, and more importantly, equipment cost.

Much of analytical TEM is based on elemental analysis of core-shell ionizations and their role in electron energy-loss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDS). In these techniques, integrals of the primary or secondary ionization signals (typically over many tens of eV in energy) are used to measure and map the elemental composition of probed sample areas.

In contrast, present-day STEM EELS systems are able to reveal spectral details with resolution in the range 0.1-1.0 eV. This means that EELS provides access to electronic structure and response information that goes beyond the simple elemental composition information of the integrated core-loss signals.

HyperionTM is a newly developed high-performance ion source that significantly advances the capabilities of many ion beam techniques used by material scientists and engineers. Hyperion has been developed to provide focused beams as small as 10 nm, beam currents up to several micro-Amps, and a broad range of ion species that include He+, O2+, Xe+ and H3+.

Focused ion beam (FIB) instrumentation has proven to be extremely useful for preparing cross-sectional samples for transmission electron microscopy (TEM) investigations. The two most widely used methods involve milling a trench on either side of an electron-transparent window: the “H-bar” and the “lift-out” methods [1]. Although these two methods are very powerful in their versatility and ability to make site-specific TEM samples, they rely on using a sacrificial layer to protect the surface of the sample as well as the removal of a relatively large amount of material, depending on the size of the initial sample. In this article we describe a technique for making thin film cross-sections with the FIB, known as Shadow FIBing, that does not require the use of a sacrificial layer or long milling times [2].

Every spring, I teach a one-semester, graduate-level course on materials transmission electron microscopy (TEM). Thanks to the explosion of interest in nanotechnology, what was once a course primarily for metallurgists on imaging crystallographic defects and x-ray microanalysis now attracts a much broader audience. I have had students in the course from almost all the engineering departments at UW Madison (materials, chemical, mechanical, electrical, civil), from the basic sciences (physics, chemistry, geology), and from other departments (including one from Food Science!). The enrollment in the concurrent laboratory class on TEM operation is similar.

This diverse student body has two consequences. First, the students' background knowledge varies widely. Some have already taken a materials characterization course that included some TEM, but others barely know what a crystal structure is.

Biological composites have attracted increasing interest because of their outstanding properties and versatility. Crustaceans are interesting models for the study of these materials. They have a mineralized exoskeleton (cuticle) that provides support and protection. The structural organization of the cuticle is ubiquitous in arthropods: a hierarchically structured organic matrix composed of planes of parallel chitin-protein fibers that are stacked in a twisted plywood organization [1] associated with various amounts of minerals. The land-dwelling crustacean Porcellioscaber (P. scaber) is a well-established model organism for the study of the bio-mineralization processes [2, 3].

Efforts to understand the basic mechanisms of angiogenesis, that is, the formation of new blood vessels from existing vasculature, have been limited by the methods that are currently used to measure vessel growth. Although in vivo assays provide the best environment in which to track angiogenesis, inherent difficulties in obtaining reproducible data limit the power of this approach. Limitations include: environmental variations between experimental animals, induction of inflammatory responses by surgical methods, and labor-intensive blood vessel quantification procedures. A better assay would measure vessel growth in one animal at multiple time points and would focus on minimization of artifacts induced by experimental manipulation.

Dip Pen Nanolithography (DPN) is a scanning probe lithography technique where an atomic force microscope tip is used to transfer molecules to a surface via a solvent meniscus. This technique allows surface patterning on scales of under 100 nanometres. DPN is the nanotechnology analog of the dip pen (also called the quill pen), where the tip of an atomic force microscope cantilever acts as a “pen,” which is coated with a chemical compound or mixture acting as an “ink,” and put in contact with a substrate, the “paper.”

DPN enables direct deposition of nanoscale materials onto a substrate in a flexible manner. The vehicle for deposition can include pyramidal scanning probe microscope tips, hollow tips, and even tips on thermally actuated cantilevers. Recent advances have demonstrated massively parallel patterning using two-dimensional arrays of 55,000 tips, depicted below. Applications of this technology currently range through chemistry, materials science, and the life sciences, and include such work as ultra high density biological nanoarrays, additive photomask repair, and brand protection for pharmaceuticals.

A unique class of impurity-based quasi-liquid films has been widely observed at free surfaces, grain boundaries (GBs), and hetero-phase interfaces in ceramic and metallic materials (Figure 1). These nanometer-thick interfacial films can be alternatively understood to be: (a) quasi-liquid layers that adopt an “equilibrium” thickness in response to a balance of attractive and repulsive interfacial forces (in a high-temperature colloidal theory) or (b) multilayer adsorbates with thickness and average composition set by bulk dopant activities [1–2]. In several model binary systems, such quasi-liquid, interfacial films are found to be thermodynamically stable well below the bulk solidus lines, provoking analogies to the simpler interfacial phenomena of premelting in unary systems [3] and prewetting in binary de-mixed liquids [4]. These interfacial films exhibit structures and compositions that are neither observed nor stable as bulk phases, as well as transport, mechanical, and physical properties that are markedly different from bulk phases.

In light microscopy, dark field and interference contrast are widely used for examination of transparent specimens. These methods both suffer from various limitations when photomicrographs have to be taken from fine details, especially in three-dimensional specimens requiring a large depth of field.

In common dark field illumination, the condenser either is not equipped with an aperture diaphragm, or an existing condenser diaphragm has to remain in the wide-open position. Thus, the depth of field is lower than in bright field images. Moreover, dark field imaging is associated with marginal blooming, especially in linear structures exhibiting with large differences in phase or density (e.g. cell walls, edges in crystals and other mineralogical material).

A disruptive innovation in how scanning electron microscopes (SEMs) are designed and built has produced a low cost, but highperformance compact field emission SEM. By leveraging silicon processing technologies, Novelx has miniaturized the core technology inside a SEM. This miniaturization enabled the design of an all-electrostatic electron beam column that when coupled with a thermal field emission (TFE) electron source is optimized for lowvoltage imaging and sub-10nm resolution. In addition, the mySEM© has a quad-segmented microchannel plate (MCP) detector that has a topographic imaging mode that is capable of electron channeling contrast imaging (ECCI) at low imaging voltages. These capabilities were previously only available in high-end and much larger sized field emission SEMs outfitted with additional detectors. The Novelx mySEM show in Fig. 1 is now being used in material science and life science applications for characterizing a variety of energy sensitive nanomaterials, biomaterials and thin films.

Good sample preparation is essential for acquiring successful electron backscattered diffraction (EBSD) patterns in the SEM. Mechanical polishing to obtain the required surface quality with minimal sub-surface defects and deformation that does not interfere with the quality of the diffraction data is, more often than not, an art form. Special polishing techniques, such as low force lapping fixtures, electrochemical-mechanical polishing, and vibratory polishing, have been used to minimize the sub-surface damage, but have not eliminated it. Ion polishing has been used to reduce the damage layer further. However, the commercially available ion systems suffer several drawbacks, including: 1) small area treatment (≤ 1 cm) 2) decreasing beam current density with accelerating voltages, and 3) the inability to process non-conducting samples. Barna and Pecz have shown that at 3 keV with an incident angle of 5° relative to the surface, approximately 25 nm of ion damage occurs in Si and GaAs, but at 250 eV, there is less than 1 nm of amorphization of the surface. They also showed that a glancing angle across the surface is essential for removing topographic features. The ion guns that have been available for ion polishing and ion etching of SEM samples typically cannot operate effectively below 3 keV because of the low current density.

It is well known that there is a profitable cooperation between archaeology and the scientific disciplines of chemistry, geology, biology, and physics with the aim of producing better interpretations of archaeological materials. This field of science is known as archaeometry. Two main goals of archaeometry are to analyze and characterize historic objects to preserve them and to investigate the knowledge and skills required to fabricate them. This latter information is essential in the evaluation of cultural and technological aspects of past societies and to further understand the transference of technological knowledge through time periods and geographical contexts.

Microwave-assisted processing of biological specimens, from its inception, has been a methodology that promised time savings over conventional processing methods [1]. It has taken almost 30 years to define and control the significant variables associated with microwave processing [2]. Recent research combined with improved technology have helped in the identification and control of the experimental variables associated with microwave-assisted processing [2–7]. Stated simply they are: (1) constant sample temperature control in conjunction with continuous microwave irradiation [3–4], (2) control of wattage in the microwave device (the ability to control microwave power in the same manner as a dimmer switch controls a light) [2–5], and (3) energy uniformity in the microwave cavity [3–4].