Fluctuation microscopy is the enigmatic name given to an otherwise straightforward technique for studying medium range order in highly disordered materials. By medium range, we mean atomic ordering at length scales within the range 0.5-2.0 nm, where traditional imaging and diffraction techniques have the most difficulty detecting structural correlations in amorphous materials. Puzzlement over fluctuation microscopy generally arises not because of the "microscopy" part of the name, but because of the "fluctuation" part. What, exactly, is fluctuating? And, why does it fluctuate?

The fluctuations are simply the variations in scattering between small sub-volumes within a thin sample. These are usually not timevarying fluctuations (although they could be), but instead they are the position-varying fluctuations in local diffraction.

In the previous article (part 1), the procedure for setting the computer screen was discussed by using Adobe Gamma in the control panel. Adobe Gamma is also found on the drive where the system is installed. Click on Program Files > Common Files > Adobe > Calibration > Adobe Gamma.cpl. Double click to launch the gamma control wizard. On a Macintosh the Apple Calibration Utility can be used.

The merits of using ink jet printers were also discussed. Merits include low cost (though costs escalate with the purchase of cartridges), high quality prints from any application, and the technology of a fine spray with higher image resolution as opposed to a laser jet printer and Tektronix Phasers (these printers separate images into dots at compromised resolutions).

Color has historically seen limited use in metallography, mainly due to the cost of film and prints and the difficulty and cost of reproducing images in publications. However, with the growth of digital imaging, capturing color images is much simpler and cheaper. Also, printing images in color is inexpensive for in-house reports, and can be distributed cheaply on CDs, although reproduction in journals is still expensive. Color does have many advantages over black and white. First, the human eye is sensitive to only about forty shades of gray from white to black, but is sensitive to a vast number of colors. Tint etchants reveal features in the microstructure that often cannot be revealed using standard black and white etchants.

Although many samples in electron microscopy are phase objects, as in the case of optical microscopy, we cannot directly measure phase modulation by microscopy. Dennis Gabor proposed a technique called in-line holography to record both amplitude and phase information at the rather early stage of electron microscopy (in 1947). With the development of highly coherent field emission electron sources, another type of holography, off-axis holography, became available for electrons. However, holography cannot be applied to general cases, since there is a requirement for a vacuum region where the reference wave passes through.

All living creatures, including humans, are made of cells. The majority of life forms exist as single cells that perform all functions to continue independent life. Some cell structures, cell organelles and particularly bacteria and viruses are commonly too small to be fully observed with an optical microscope. Therefore, an electron microscope is required. Since samples examined with an electron microscope are exposed to very high vacuum, it is impossible to view living cells. The sample preparation for electron microscopy requires that living cells be killed, frozen, dehydrated, and impregnated with heavy metals.

Bacteria contain a wealth of mechanisms that organize their internal and external components into a highly polar structure, frequently with distinctive shape, and constrain certain metabolic functions to particular parts of the cell. For example, cell division generally takes place at the middle of the cell, and a host of interacting proteins are involved in ensuring that the division site is positioned properly. Thus, in spite of the lingering perception among some scientists that these cells are simply bags of freely diffusing enzymes, there is much to be learned from ultrastructural studies. Light microscopy has given evidence of cytoskeletal components that presumably establish and maintain cell shape and participate in cell division.

An earlier article in Microscopy Today, "Rediscovery of Darkfield Dispersion Staining while Building a Universal Student Microscope," noted that the darkfield inserts were made for Peter Cooke (MICA, Chicago, IL) to be used to teach high resolution dispersion staining in his advanced microscopy classes using Olympus BH2 microscopes and the Olympus 1.25 NA Abbe condenser. Peter has asked whether there is a better condenser design that will reduce the time and effort needed to switch between brightfield and darkfield.

Viral infections can be focal and therefore difficult to find by electron microscopy. In addition to sampling limitations, sometimes the only specimen available for examination is tissue that has already been prepared for light microscopy (LM). We have diagnosed a papovavirus infection in skin by embedding hematoxylin and eosin (H&E)-stained sections for ultrathin sectioning and transmission electron microscopy (TEM).

Immunosuppressed patients can have unusual infections or infections in unusual locations. These agents may be difficult to identify due to their not being suspected and incorrect tests being ordered.

Production of crude oil is generally accompanied by several other product phases, namely water, gas and solids. Pressure drops across chokes, concomitant gas evolution (due to pressure drops) and turbulence caused by various pipeline configurations can create difficult-to-resolve emulsions. Natural crude oil surfactants and solids exacerbate the problem further by migrating to the newly created oil-water interface and stabilizing the unwanted emulsions. Once the fluids arrive at the production facilities, a variety of vessels are employed to separate the oil, gas and water. Depending on the wettability of the solids, they will exit via one or both of the liquid phases. In a worse case scenario, the solids will accumulate at the oil-water interface.

This guide provides an introduction to calibrating transmission electron microscopes (TEM's) and scanning transmission electron microscopes (STEM's), as well as (S) TEM's - instruments that can perform in both modes. While not technically correct, all these instruments will be collectively referred to as TEM's for the rest of this Guide. As with most things, there is a wealth of interesting facts and insights to be discovered by exploring this topic further. More detailed explanations can be found in the classic monographs by Edington or in textbooks such as Williams and Carter.

Fundamental research within the scope of cell and structural biology as well as nano-technology is increasingly focusing on unraveling interactive biological and biochemical processes and pathways at the macromolecular level. For this, high resolution transmission electron microscopy (TEM) is indispensable. Of paramount importance is the three-dimensional visualization of macromolecular structures and molecular machines in their native hydrated state. Their physical fixation within ultra-thin vitrified ice layers is the crucial starting point.

Materials research, biomedical research, and semiconductor manufacturing can all benefit from nondestructive, high-resolution methods of analysis. As most materials are heterogeneous, it is important to not only acquire high resolution topographic information, but also to identify the chemical composition of samples. A combination of high resolution microscopy with chemically sensitive spectroscopy combined in one instrument allows the detailed characterization of samples with different analytical techniques. When individual instruments are used, returning to a previously surveyed sample area can be very time consuming if not impossible without surface markers.

When preparing specimens for TEM analysis, it is important that specimens routinely be large enough to manipulate with forceps and cohesive enough to hold together as a single entity. However, anyone working in EM will have some specimens that are too small to see or centrifuged pellets that easily fall apart during processing. These specimens can easily be lost during a routine dehydration and embedding protocol and therefore cannot be sectioned. If the specimen to be processed is too small to handle or will fall apart, then precautions can be taken to ensure that it can be safely embedded for sectioning. Although this article is written with the main focus on biomedical electron microscopy, these handling protocols can be applied to any small specimens that have to be thin sectioned.

When electrons pass through a material, they can pass through without losing energy such as elastically scattered electrons or they can lose or gain energy by inelastically scattering with the material's electrons. The elastically scattered electrons have been used in the simulations of lattice images, which are used to help determine the atomic structure of materials. Inelastically scattered electrons were ignored in the simulations because it was believed that they did not have the required coherence to interfere with themselves and they contributed only to the background intensity. Recently though, a great deal of interest has been generated in knowing whether the inelastically scattered electrons can also contribute to the lattice images in order to help explain the Stobbs factor [1], where the contrast in the lattice images is often three or more times less than the theory predicts. The Stobbs factor makes it difficult, if not impossible, to establish quantitative values to high-resolution lattice images.

Since the publication of the use of a dicing saw for TEM sample preparation, several analytical labs have adopted this method as standard practice for site-specific cross section and plan view samples. In this article, we would like to provide additional practical details of these procedures, and describe several extensions, including useful notes on batch processing, preparing samples with an area of interest very close to the sample edge, and a Focused Ion Beam (FIB)-compatible sample holder. We present an unusual amount of detail in these processes to show some of the evolution of the method since its introduction and to allow others to easily reproduce these results.

Sample preparation for Transmission Electron Microscopy (TEM) is usually performed such that the final sample orientation is either a cross section or a plan view of the bulk material, as shown schematically in Figure 1. The object of any sample preparation technique, for either of these two orientations, is to thin a selected volume of the sample from its initial bulk state to electron transparency, ~ 100nm thick. In doing so, the final sample must be mechanically stable, vacuum compatible, and, most of all, unchanged from the initial bulk material. Many techniques have been used to achieve this goal: cleaving, sawing, mechanical polishing, chemical etching, ion milling, focused ion beam (FIB) milling, and many others.

The primary responsibility of the clinical electron microscopy laboratory is to produce, and deliver to the pathologist or clinician, high quality ultrastructural images of pathological specimens in a timely manner. In order to facilitate this process, medical EM labs are converting from traditional chemical processing of photographic film and paper to the acquisition and transmission of electron images by computer. Ideally the electron microscope would incorporate a digital camera in the column to collect the images, thus bypassing chemical processing of film and paper altogether. However, due mainly to the high cost of the in-column cameras, many labs continue to use film, but replace the time consuming enlarging/printing in the darkroom with digital techniques at the computer.

"The most advantageous feature of the ex-situ lift out method is throughput."

A great deal of emphasis is placed on "throughput" in the microprocessor industry. Wafer sizes are getting larger and the costs of building them have increased astronomically. The transmission electron microscope (TEM) has become the essential tool for examining current microprocessor products. The TEM can only be effective if it has properly prepared specimens to put into it. In order to achieve the highest specimen preparation spatial resolution, the microprocessor industry has turned to focused ion beam (FIB) tools, either single or dual column, for TEM specimen preparation in applications ranging from process control to failure analysis, and on to semiconductor device metrology.

The majority of human pathogens cause disease in a biofilm lifestyle. Biofilms are communities of cells that remain attached to a foreign surface and to each other. The organisms secrete and become embedded in an extracellular polysaccharide matrix. Biofilm infections occur most commonly on implanted medical devices and are quite recalcitrant to antimicrobial therapy. The most common device involved in these infections is the intravenous catheter. More than 5 million intravenous catheters are inserted into patients in the United States annually of which up to 10% become infected with biofilm producing pathogens.

In 1965, Gordon Moore forecast that the microprocessor industry would continually scale to smaller feature sizes and the number of transistors would double every 18 months. Scaling below the 100nm node, combined with the implementation of copper and low dielectric constant insulators to increase the processor speed, has produced the situation in which SEM inspection no longer offers suitable resolution to image key artifacts and structures. The transmission electron microscope (TEM), once considered more of a development tool, is now in the forefront for process control and failure analysis, especially for measurements such as the thickness of semiconductor device non-planar barrier and seed layers