In order to get high resolution images from any scanning beam microscope one must be able to produce a sufficiently small probe, have a small interaction volume in the substrate and have an abundance of information-rich particles to collect to create the image. A typical scanning electron microscope is able to meet all of these requirements to some degree. However, a helium ion microscope based on a Gas Field Ion Source (GFIS) has significant advantages over the SEM in all three categories.

With the mapping of the mouse genome, the growing sophistication in transgenic sciences creating complex mouse models of disease, and the demand to study disease in vivo, there has been a corresponding increase in the demand for and development of preclinical imaging modalities. Clinical ultrasound operating in the 2-12 MHz range is a well established clinical imaging modality, accounting for more than one-third of all imaging procedures performed in North America. The simplicity, ease of use, speed, and safety of ultrasound have led to its significant role in diagnosis, treatment assessment, follow-up, and guidance of therapy in clinical applications. Ultrasound imaging is used routinely in its B-Mode imaging mode to report on soft tissue structures. It's also used in its Doppler modes for the measurement of blood velocity in fast-flowing targets such as the cardiovascular system, in slow-flowing applications such as quantifying blood flow and in vascular architectures within tumors.

Low voltage field emission SEM (i.e., operated at several hundred volts to 5 kV), offering advantages in surface imaging due to reduced beam penetration,1 was found to be particularly useful in the investigation of uncoated, fine, geological materials down to the nano-scale. Here are four examples to highlight projects being conducted in our FESEM facility.

Kaolinite is one of the most important industrial minerals. Information about its surface properties and cation exchange capacity are important in both ore processing and applications of the clay mineral.2 Low voltage (LV) secondary electron (SE) imaging (Fig. la) reveals detailed (001) surface features of kaolinite, which are not obtainable with high voltage SEM (Fig. lb) and TEM. Kaolinite is highly sensitive to beam damage and can show obvious degradation after just a few seconds under the TEM beam. However, microtomed TEM samples maybe easily examined under LV SEM to show their thickness perpendicular to (001) and cross section features. Such information allows easy access to direct surface area determination of clay minerals.

Polymers are viscoelastic materials that can often deform during microtome sectioning. Similar to plastic embedded biological materials, many methods have been developed over the years to not only improve the image contrast of these materials but also to harden the material for improved sectioning during microtomy. Even with these improvements, a common artifact, compression, during the sectioning of this class of materials remains problematic.

Compression is caused by several factors: hardness of the sample, embedding media, wedge angle of the knife, interaction between the diamond and sample surface, section thickness and cutting speed. It has been found that reducing the knife angle from 45º to 35° leads to a reduction in compression. Recent efforts to further reduce the compression of ultra-thin sections have led to the invention of an oscillating diamond knife.

Electron microscopy and x-ray microanalysis are common techniques in many research, development and quality labs (Garratt-Reed and Bell, 2002; Goldstein et al., 2003). As systems become ever more automated, as well as easy to use, new applications for the use of these systems develop and expand. One such application is particle analysis. These “particles” might be small loose fragments that can be applied to a conductive adhesive or a filter, embedded in epoxy and polished, or they can be inclusions within collect an EDS spectrum.

Traditionally, type specimens have not been deposited for most unicellular algae. There are few known holotypes and those that are available are often of limited use. Samples from taxa described before 1900 commonly consist of materials dried down on herbarium sheets or mica. Dried algal samples can be rehydrated, but preservation is very poor and little structure can be determined from them. Whether wet or dry, the amount of information that can be derived from such samples using conventional light microscopy is limited.

Fluorescence microscopy has experienced tremendous growth over the past four decades and has facilitated major advancements in science. The classic fluorescent techniques (which include epifluorescence and confocal) allow researchers to selectively observe labeled structures with great clarity and consistency. Historically, biological scientists have been the most prolific users of fluorescence imaging. However, growing numbers of nano-materials research initiatives are now incorporating quantum dots and other fluorescent labels into their imaging protocols. This is especially true in areas where nano-materials and biological research is overlapping such as drug delivery. This nano-bio convergence, along with other advancements, has generated the need to observe highly dynamic events involving labeled and unlabeled structures in real time.

SEM observation of a specimen cross section can provide important information for research and development as well as failure analysis. In most cases, surface observation alone cannot provide information concerning the cross sectional structure of granular materials, layered materials, fibrous materials, and powders. Preparing highly-polished cross sections of these materials is both a science and an art.

Typically, a cross section is prepared using mechanical means like conventional mechanical polishing methods or a microtome. The sample is first embedded in a holder or device, and then polished to achieve a flat cross section. In some cases, a staining procedure is used to highlight a specific component of the sample. Such methods can be lengthy procedures that require a great deal of skill, and can introduce artifacts into soft materials, deform the material around voids, or compress layers of soft and hard materials in composite samples. Mechanical polishing can miss fine details such as the presence of hairline cracks, and present a challenge to water-soluble phases.

The ultimate performance of high resolution electron microscopes depends on a variety of factors including the stability of the temperature surrounding the instrument. Variations in temperature can cause drift of the specimen, the microscope electronics, and the mechanical tolerances in components such as microscope lenses and scan coils. For high resolution work or for image reconstruction that requires acquisition of multiple exposures over extended periods of time, it is critical that the temperature variations be strictly controlled. A typical manufacturer's specification for temperature stability of a room housing a transmission electron microscope (TEM) is a tolerance of <0.5°C/hour with fluctuations of <0.05°C/minute. While modern instrumentation rooms are carefully designed to maintain stable environmental conditions (O'Keefe etal, 2004), it seems unlikely that these stringent specifications are routinely met in practice when the microscope is in operation and personnel are present in the room.

Perfusion, washing out blood and using the open vascular channel to infuse fixative, is the standard first step of preparing animal tissues for later examination under a microscope. The rapid and homogeneous fixation resulting provides an advantage over immersion fixation that is usually used for biopsy and clinical tissue samples (Cammermeyer, 1960, Garman, 1990). A disadvantage is that brain tissue, and probably other soft tissues, prepared by perfusion has no retained extracellular space post perfusion, although living brain has about 20% extracellular space (discussed in Cragg, 1980). Perfused brain is also about 20% shrunk in whole organ volume from living brain size. The shrinkage is uneven, and distorts the relationships of structures. It is possible to avoid the organ shrinkage and distortion that follows a traditional perfusion, with little additional effort. This is a discussion of how to optimize your perfusion results, why the shrinkage occurs, and how it can be avoided.

The Focused Ion Beam (FIB) instrument, originally designed for semiconductor circuit modification and repair, has found considerable utility as a tool for specimen preparation in several microscopy disciplines and for micromachining small parts. Essentially, a FIB makes very small and precise cuts into a target sample, which implies well-controlled etch rates and close tolerances of surface finish. However, redeposition can affect etch rate and final surface topographies. This work quantifies this redeposition as it modifies yields for different parameters of etching; models the influence of redeposition as applicable to all ion beam processing; and optimizes FIB processing parameters for enhanced yields.

The present situation in Ecuador is not a happy one. You would think that this ought not to be the case. There are seven electron microscopes in the country and all of them are in working order (although with problems - see below), so why then are things not good. The problem begins with geography. Unlike most smaller countries, Ecuador is not dominated by a single large city. There are two major cities, locked in a continuous rivalry: Quito, the capital, is in the mountains, and Guayaquil, the larger city, is on the coast. They are about seven hours apart by road. Both cities have electron microscopes. Worse, however, is the fact that, in each city, the microscopes are in two different locations - making a total of four microscopy centers. This is too many and Ecuador can not support them all effectively.

Spurr low viscosity embedding medium was introduced in 1969 and used vinyl cyclohexene dioxide (VCD or ERL 4206) as the low viscosity epoxy resin together with nonenyl succinic anhydride (NSA), the flexibilizer DER 736, and the accelerator dimethylaminoethanol (DMAE or S-1). This low viscosity formulation has been used extensively for embedding plant materials, microrganisms and other difficult specimens. Over the years numerous concerns about the toxicity and carcinogenicity of VCD have been raised. VCD (Fig. 1) was finally removed from the market in 2005 and replaced with ERL 4221 (Fig. 2), a cyclic epoxide with a higher molecular weight and higher viscosity. Vendors instructed that ERL 4221 be substituted for VCD at the same rates as used in the original Spurr formulation. This direct substitution resulted in a more viscous resin and blocks were extremely hard and brittle and lacked the sectioning qualities of the original formulation. Vendors then suggested modifications to the dehydration schedule and reductions in the amount of DMAE to improve the quality of the blocks.

Recent advances in nanotechnology and nanoscience are highly dependent on our newly acquired ability to measure and manipulate individual structures on the nanoscale. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution dictated by the laws of diffraction at about 250 nanometers. This diffraction limit arises from the fact that it is impossible to focus light to a spot smaller than half its wavelength. The challenge of breaking this limit has led to the development of near-field scanning optical microscopy (NSOM).

In the three decades following Castaing's seminal thesis [1] x-ray analysis received widespread attention from research groups. By 1980, the methods and correction procedures for quantitative analysis of elements with atomic number 11 and above, using accelerating voltages between 15kV and 25kV, were well established and available in commercial instrumentation. At the time, scanning electron microscopes (SEMs) could rarely deliver high and stable beam current at much lower kV, and x-ray spectrometers had poor efficiency below lkeV so that low kV analysis received comparatively little attention.

Resource sharing has become an absolute necessity for modern scientific research because of the increasing expense and complexity of instruments, and the ever changing funding paradigm which often requires sharing of major instrumentation resource across multiple disciplines. Many universities and even large companies have set up centralized facilities to serve researchers from different departments, divisions and units to minimize expenses and maximizing usage.

Just in the materials research area alone, it is estimated that there are more than 500 mid-size facilities existing nationwide, as reported in 2005 by the Committee on Smaller Facilities of National Research Council. Resource sharing is also common in large research groups, between Principal Investigators, as well as in corporate/industrial laboratories.

Of all the analytical techniques in electron microscopy, X-ray energy dispersive spectroscopy (XEDS) is perhaps the most useful. It provides a quick identification of the elements and even with semiquantitative methods; a reasonable composition can be obtained. However, in the scanning electron microscopy (SEM), there are materials systems in which severe peak overlaps of heavier elements L and M lines cannot be easily deconvolved with lighter elements' K lines. In addition, without a sufficient overvoltage in the SEM, even identification of the heavier elements can be difficult. In the analytical electron microscope (AEM), there is always sufficient overvoltage to excite all of the elements' K-lines. However, all of the K-lines might not be able to be detected with commercially available instruments. This is illustrated in Fig.l where the maximum energy of the detector system might be set to 10, 20, or 40 keV.

Since the early 1990s EBSD (Electron Back Scatter Diffraction) has been developed to become a mature crystallographic analysis technique at the micro and nano-scale. It is applied in a SEM (Scanning Electron Microscope) on samples with a very smooth and clean surface. It provides quantitative orientations of individual grains, and by mapping a larger area, multiple grain analysis, texture, and grain boundaries can be examined. As the useful information for the EBSD technique comes from a very shallow depth in the material (10 to 20nm), it is a surface analysis technique showing lateral 2D distributions of crystal orientations.

JECP stands for a java electron crystallography project that includes a series of practical java stand-alone programs for electron diffraction/microscopy and crystallography applications. The aim of the JECP project is twofold, i) as teaching tools to show students the principles of electron diffraction/microscopy and crystallography, ii) as research tools to analyze experimental results. Although there are commercial and public domain computer programs available that allow a user to simulate electron diffraction patterns or processingHREMimages, there are always situations when we need to perform operations that are not a feature of any of the existing programs. The programs in the JECP can bemodified and extended tomeet the need in experiments.