Energy-Dispersive X-Ray Spectroscopy (ED-XRS or EDS) is a powerful and easy-to-use technique for the elemental analysis of a wide variety of materials. Most commonly, this technique is called X-Ray Fluorescence (XRF), which classically uses x-ray photon sources to excite the sample. A Scanning Electron Microscope (SEM), of course, uses electrons as the excitation source for microbeam x-ray spectroscopy together with sample imaging using characteristic x-rays and/or secondary electrons. These two XRS techniques are used independently, although often the same sample is analysed by both, to provide complementary information.

The advantages of both techniques have been reviewed several times [e.g. 1,2], SEM-EDS being more suited to imaging and microbeam quantitative compositional analysis and maps, and XRF more suited to accurate quantitative analysis, especially for trace elements, while analyzing a much larger area.

The current use of microwave technology in science creates a dichotomy. Is it the heat or is it the energy? One entire branch of science, chemistry, uses microwave energy to apply heat to a broad range of chemical processes, under pressure, to produce the desired end-products quickly and efficiently (1). The biological sciences, surgical pathology in particular, have tried to adapt the microwave oven to speed up a broad range of processes: fixation, decalcification, antigen retrieval, tissue processing for paraffin and plastic embedding, and histological staining, including special stains, immunolabeling, and in situ hybridization (2). The biologists have assumed that they are also applying heat to speed processing. However, recent improvements in the microwave suggest that the energy is the critical variable (9). We have designed fixation experiments to test the two views.

The exit electron wave from transmission electron microscopy (TEM) specimens contains both amplitude and phase information. In routine TEM imaging, only amplitude information is recorded on the recording devices (film or CCD camera) and phase information of the electron wave function normally is canceled out.

In 1947, Dennis Gabor proposed off-axis electron holography, a method of interference imaging in which the phase and amplitude components of the electron beam are obtained to correct spherical aberration of the transmission electron microscope to improve spatial resolution. In that process, the electron beam is split into the two beams: the un-scattered electron beam (i.e. the reference wave) and the image beam (or object wave) diffracted by the specimen and exiting the bottom of the specimen surface.

The recent article in this journal by O'Keefe et al provides an excellent introduction to the complexities that must be considered when embarking on the installation of a high-resolution electron microscope. It should not pass one's notice, however, that the advent of nanoscience has placed ever stricter attention on the control of vibration not just for analytical instrumentation but also for fabrication facilities. In addition to the thick isolated concrete slab-ongratie described in the above article, designs are coming into use (Fig. 1) that incorporate rigid “waffle” floor structures on closely spaced building columns and pneumatically isolated inertia slabs as are used, for example, in the subterranean portion of the NIST Advanced Metrology Laboratory. Each of these approaches might with time also find applications in the design of electron microscopy laboratories. However, each has the problem of rather high expense of construction.

It is a common practice to use microscopic images to describe the differences observed between plant tissues. The images illustrate the taxonomic characteristics of the studied species. In this work we introduce a quantitative method for conducting these analyses utilizing digitized images obtained via scanning electron microscopy (SEM) of barley leaf surfaces. The topography of the leaf surfaces of a narrow-leaf mutant and its wild type mother line was characterized, see figure 1, using the Rotated Image with Maximum Average Power Spectrum (RIMAPS) technique and the Variogram method. Spectra resulting from RIMAPS analysis allow us to identify the specimens and to distinguish between the adaxial or the abaxial side of the leaf. These results are complemented by obtaining the typical scale lengths that characterize the abaxial surfaces of both the mutant and the mother line barley leaves.

In characterization or failure analysis of new materials and semiconductor devices, the requirements for three dimensional observation and analysis are rapidly increasing. We discuss techniques for specimen preparation, three-dimensional observation, and elemental analysis of semiconductor devices that we developed using a system consisting of a dedicated focused ion beam (FIB) instrument and a scanning transmission electron microscope (STEM). The system utilizes a FIB-STEM compatible specimen holder with a specially designed rotation mechanism, which allows 360° rotation of a specimen on a conical stage (needle stub) around the ion beam axis of the FIB system and 360° rotation perpendicular to the electron beam in the STEM. A piece of sample (micro sample) is extracted from a specific-site by the micro-sampling technique and mounted on the needle stub. Instruments used in the study are the Hitachi FB-2100 FIB system with a micro-sampling attachment and the HD-2300 field emission 200kV STEM.

One problem facing owners of high-end digital microscope cameras and scanners is dealing with 12-bit TIFF format images. Because of a vagueness in the TIFF specification [1], some prograins do not load 12-bit TIFF images at all, or do not handle them gracefully. Unfortunately, Adobe Photoshop is one such program.

A common problem with 12-bit images that do load in Photoshop is that the image appears to be totally black once loaded. This occurs when the camera stores the most significant bits of the image in the lower 12-bits of a 16-bit data space. Photoshop, when converting to its internal 16-bit representation, does not scale these properly, resulting in a low contrast image. This can present problems with viewing the images, or performing any processing or measurement steps that you might desire.

Many dynamic processes do not occur in nature, science or industry in a typical TEM high (and sometimes not so high) vacuum environment. Dynamic in-situ data related to the real world need to be obtained under Controlled conditions of gas/vapor/liquid environment and temperature. In the ETEM (Environmental Transmission Electron Microscope), the specimen - but nothing much else - is shared between the chemical reactor on the horizontal axis and the vertical microscope column (Fig.1). The original Philips CM30 column is highly modified with pressure limiting apertures around the beam and multiple stages of differential pumping (Fig.2).

The One-Ångstrom Microscope (OÅM) project was established at the NCEM to produce images at sub-Ångstrom resolution (O'Keefe, 1993). The project was implemented using a Philips CM300FEG/UT with hardware modifications designed to correct objective lens three-fold astigmatism and extend information transfer to 0.8 Ångstrom (O'Keefe et al., 2001a). A Gatan image filter (GIFTM) was used to bring the image magnification to more than three million times at the CCD camera to provide adequate real-space sampling.

Phase-contrast imaging in the HRTEM produces images with peaks at atom positions by extracting the spatial distribution of the relative phase from the electron wave. Usually, the electron wave is imaged by direct interference of diffracted beams at optimum focus (Scherzer, 1949).

A recent thread on the MSA list server about problems with image formats (in this case TIF or TIFF) showed, that there is a bit of confusion in the microscopy community about the best file format for digital images. I will try to shed a bit of light onto this problem.

Digital images are at the core a large array of numbers. One number per pixel for b/w images, 3 numbers per pixel for color images. The simplest file format consists of 2 numbers that define the width and height of the image, and then just a listing of numbers for each pixel. By using the right conventions, the image can be recreated from a data file like mis. In essence, this is the format of a bitmap image (BMP), and other formats derived from it.

Technical publications sometimes include scanning electron images to characterize a microstructure, when the relevant structure could have very easily been illustrated using a simple light micrograph. When should one use a light generated image? What are the advantages/disadvantages of an electron generated image, particularly; one generated using a scanning electron microscope (SEM)? While there is some overlap in the capabilities of these imaging systems; in general, they are complementary tools, each with their own uses. Standards under the jurisdiction of ASTM Committee E4 on Metallography offer guidance to both new and experienced users of both investigative techniques.

Yeasts are single-celled eukaryotic microorganisms (generally about 5 to 10 microns in diameter) that divide by a budding process and are classified with the fungi. Yeast cells are ubiquitous in our environment and can be found on plants and in soil and water. Yeasts have considerable importance Ln industrial and agricultural settings, Saccharomyces cerevisiae (Figure 1) is also known as “bakers yeast” or “brewers yeast.” Specific strains of yeast are used to make pastries, bread, beer, ale, wine, distilled spirits, and industrial alcohol. In the paper industry, Candida utilis is used to break down die sugars from processed wood pulp. Yeast cells are also nutritious. In some societies, “cloudy” beer (containing yeast cells) provides essential B vitamins, minerals, and amino acids.

Delamination is defined as separation into constituent thin layers (lamellae). Glass delamination, which can have several causes, could then be defined as a separation of surface glass, as from a vial, into thin layers resulting in a flaky appearance. Pitting could be related to, or even be the initial stages of glass delamination; data will be provided that supports a relationship. The presence of glass delamination is a serious concern to the pharmaceutical industry, not only because suspended glass flakes in any pharmaceutical product poses an obvious health hazard, but because of regulatory quality control standards, and medical profession requirements.

There are, of course, many ways of addressing the problem of glass delamination in pharmaceutical vials, including looking into the relationship of chemical nature of vial contents to degree of delamination and conditions of vial manufacture. Specialized lighting and imaging methods are other research approaches.

The ability to print what is seen on a computer screen so that it matches a printer (color matching) confounds even the most experienced. That's because computer monitors, even from the same manufacturer, cannot be made to display colors identically. Also, printers from different manufacturers contain several “libraries” of replacement colors in order to replace pixel values in an image from light (red, green and blue: RGB) to the primary colors for pigments (cyan, magenta, yellow and black: CMYK). Cameras use the primary colors for light and that is the way the digital image is stored and displayed; printers use the primary colors for pigments in ink. Thus, a printer must have a pixel by pixel list of replacement CMYK values for every RGB value in any image for that manufacturer's idea of faithful color matching.

The technique of embedding TEM specimens in polymer resins for subsequent ultra-thin sectioning is well established. For geological materials, this protocol prevents specimen damage introduced by ion beam thinning, and is often used for preparing friable or porous materials. However, traditional polymer-based embedding resins may introduce organic carbon contaminants, producing artifacts in carbon analyses. Thus, a carbon-free substitute for traditional embedding media is required to ensure accurate carbon analyses of embedded and ultramicrotomed TEM specimens.

A suitable technique that was first reported by Bradley is to embed specimens in pure sultur. In this technique, the specimen is immersed in liquid sulfur, the liquid is solidified, and the resulting block is ultramicrotomed as with traditional resins. Since sulfur has a high vapor pressure at room temperature, the ultra-thin sections are then placed in a separate vacuum chamber to sublimate the sulfur, yielding TEM specimens free of any embedding medium.

Historically, one of the first jobs performed by the new instrument called a “transmission electron microscope” (TEM) was the determination of particle size and distribution. Particle size is still important in controlling the properties of sub-micron and nanoparticle products. The width and average particle size can determine the light scattering properties of a pigment like TiO2, for example. The cost of installing a TEM facility and TEM sample preparation issues makes TEM analysis expensive and labor intensive. So decades ago, nitrogen BET (Brunauer, Emmett, and Teller) surface area determinations were used on sub-micron powders and pigments to minimize these costs. The BET measurement of the exposed external surface area of a material is made by detecting and measuring the amount of nitrogen given off after absorption, assuming one atomic layer of nitrogen is adsorbed.

Chromatic aberrations are defects in an imaging system caused by the fact that different wavelengths or colors of light are refracted by different amounts. There are two types of chromatic aberration: longitudinal and lateral. Longitudinal Chromatic Aberration arises when a lens fails to focus various colors sharply in the same plane. If white light is used, the resulting image will be unsharp due to the different focal points of its component colors. Some colors will be in focus (and therefore sharp) and other colors will be out of focus. Lateral Chromatic Aberration results in a lateral shift of the different color components of an image as a single lens with a fixed refractive index will disperse each color by different amounts. This results in color stripes at slightly different magnifications, much like a rainbow, around hard edges and a general softening or decrease in resolution in all areas.

Electron tomography using transmission electron microscopy (TEM) and related techniques (e.g., scanning transmission electron microscopy (STEM) or energy filtered TEM (EFTEM)) allow for 3-D microstructural and elemental mapping of specimens, and has been used successfully in the biological sciences where mass-thickness contrast dominates these mostly amorphous materials. Z-contrast STEM imaging via high angle annular dark field (HAADF) tomography has also been used successfully in the physical sciences. STEM, EFTEM, and holography tomography are more useful techniques for crystalline materials, since diffraction contrast in conventional TEM images can hinder image reconstruction. Typical tomography routines utilize conventional electron transparent foils, whereby the dimensions of the specimen perpendicular to the electron beam may be orders of magnitude greater than the specimen thickness parallel to the electron beam. Using this conventional specimen geometry, the effective specimen thickness increases as the specimen is tilted through the ± 70 degrees necessary for the tomographic acquisition process.

After carbon nanotubes (CNT) were discovered in 1991, many applications have been proposed that utilize their extraordinary electrical and mechanical properties. One application is as tips for scanning probe microscopy where CNTs offer several advantages including high resolution and the capability to image deep, narrow structures. A recent study of CNT scanning probes for atomic force microscopy (AFM) in semiconductor surface science concluded that an AFM with CNT tips has immense potential as a surface characterization tool in integrated circuit manufacture. Previously researchers had to construct their own CNT probes, but recently CNT AFM probes have become commercially available.

Carbon nanotubes (sometimes called buckytubes) are closed seamless shells ot graphitic carbon typically one to tens of nanometers in diameter and several micrometers long. The structure of a closed-dome single-walled nanotube is illustrated in Figure 1. Carbon nanotube probe tips offer several advantages.

The use of microwave energy to assist in the processing of biological tissues for microscopy has generated significant interest in recent years. Microwave (MW) processing has been used to prepare tissues for light microscopy (Carranza et al. 1990 [using parasite tissues]; van Dorp et al. 1995; Davis et al. 1997; Izumi et al 2000; and Rohr et al. 2001), as well as for electron microscopy (Kasa et al. 1982; Hopwood et al. 1984; Leong et al. 1985; Kang et al, 1991 [using parasite tissues]; Heumann 1992; Wagenaar et al. 1993; Login and Dvorak 1993; Giberson and Demaree 1995; Madden and Miriam 1997; Giberson et al. 1997; Morin et al. 1997; Petrali and Mills 1999; Massa and Arana-Chavez 2000; Hernandez and Guillen 2000 [using parasite tissues]; Demaree 2001; Giberson 2001).

Most reports, particularly those published by manufacturers of microwave ovens, have shown positive results regarding tissue ultrastructure, however discussion continues on possible mechanisms of preservation by the use of MW technology.