When an electron beam interacts with a solid target a number of interactions occur which produce electrons, x-rays and light. Typically in an electron microprobe analyser (EPMA) both the electron and x-ray signals are collected for analysis and imaging. However, if the EPMA is equipped with an optical spectrometer then all three signals can be collected. Commonly, the optical or CathodoLuminescence (CL) spectrometer is a monochromator type and can only collect a single frequency or small range of frequencies at a time. Simultaneous collection of the complete visible spectrum is not possible. The collection optics associated with the spectrometer often must be moved into place to start collection, this then obscures the other detectors and prevents simultaneous collection. At CSIRO Minerals an optical spectrometer has been integrated into a JEOL 8900R EPMA and allows simultaneous collect of all light, x-rays and electron signals. This form of mapping, termed Holistic Mapping, has significant advantages over traditional mapping in that it removes the need to have a priori knowledge about what the important frequencies are that will provide the solution to the problem at hand.

Oil immersion lenses have been used in optical microscopy for a long time. The light’s wavelength is decreased by the oil’s index of refraction n and this reduces the minimum spot size. Additionally, the oil medium allows a larger collection angle, thereby increasing the numerical aperture. The SIL is based on the same principle, but offers more flexibility because the higher index material is solid. in particular, SILs can be deployed in cryogenic environments. Using a hemispherical glass the spatial resolution is improved by a factor n with respect to the resolution obtained with the microscope’s objective lens alone. The improvement factor is equal to n2 for truncated spheres.

As shown in Fig. 1, the hemisphere SIL is in contact with the sample and does not affect the position of the focal plane. The focused rays from the objective strike the lens at normal incidence, so that no refraction takes place.

The production of semiconductor chips and packaging materials involves the use of a wide array of materials, from solvents and polymers, to photoresists, to metal and dielectric layers, to conductive and thermal pastes. Characterization of these materials, both in raw form and as formulated for in-process use, is integral to successful use of them in microelectronic manufacturing. Physical and chemical analytical techniques are employed to determine parameters such as composition, cure state, and interface chemistry. More often than not, it is the successful combination of complementary analyses which provide the complete understanding of material parameters needed.

This paper reports the use of Raman microprobe spectroscopy as a characterization technique for microelectronic materials. Several examples will be given, illustrating the type of information which can be obtained and the complementary use of Raman spectroscopy in conjunction with other analytical techniques such as Fourier transform infrared spectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS) to characterize microelectronic packaging materials and organic materials used in semiconductor manufacture.

FTIR and Raman microanalysis may be used as a powerful combination to determine the identity, and hence infer the source, of contaminant particles which diminish yields of semiconductor components and devices. The complimentarity of these techniques arises from the underlying spectroscopic selection rules.

Vibrational spectroscopic techniques are commonly used to characterize the molecular structure of bulk organic materials. These bulk materials typically represent purified fractions of components which may be further investigated with various classical instrumental techniques such as Differential Scanning Calorimetry (DSC), Nuclear Magnetic Resonance (NMR) spectroscopy, UV-Vis spectroscopy. However, these classical technique may have limited value for the interrogation of small impure particles or materials of limited quantity(ng.).

Elemental techniques such as Scanning Electron Microscopy coupled to Energy Dispersive Spectroscopy are enhanced by the specificity of FTIR Microprobe Spectroscopy and Raman Microprobe Spectroscopy which are now used in process laboratories to characterize and identify particulate and thin film residues with the intent of device yield enhancement.

The analytical needs in semiconductor technology have developed according to the increasing demands imposed by size and complexity. Early optical visualization, localization and counting of ever-smaller particles extended almost immediately to their morphological and chemical characterization. This evolution was due to the shifting balance from quality control to root-cause analysis. Root-cause analysis requires a deeper analytical approach to the characterization of the observed defects. Atomic composition may suffice at times as an answer. However, this information may be ambiguous and inadequate. Therefore, deeper chemical information regarding molecular structure and crystallinity of the particle is now required.

The analytical capabilities and versatilities associated with optical spectroscopy inevitably have led to their increasing applications in the semiconductor industry. in particular, the complementary Infrared and Raman spectroscopies on one hand provide huge insight into the molecular structure and crystallinity of the particles, on the other hand Raman and Photoluminescence (PL) spectroscopies are easily coupled to systems providing particle visualization and localization.

With the advent of easy to use analytical and research instrumentation the Raman technique has finally become accepted and is now widely used by pharmaceutical and biomedical companies. Applications range from simple polymorphic crystal form and protein studies to Raman mapping studies of active/excipient distribution and association that give a better understanding of drug dissolution and effectiveness. Examples of such work will be discussed.

More recently with the growing confidence in the power of Raman spectroscopy pharmaceutical and biomedical companies have teamed with instrument companies to develop specific Raman tools. One example of this is the development of a Raman mutiwell/microarray analyzer. High Throughput screening (HTS) is the latest approach in analytical technology. Used particularly in the pharmaceutical industry and in the life science laboratories, this method enables systematic screening of drug design and high throughput analysis of batch production. Combinatorial chemistry is a statistical approach in synthetic chemistry.

Many drugs are known to crystallize in different polymorphic forms or as solvates. Solubility, melting point, density, hardness, optical properties, vapor pressure, and a host of other physical properties may all vary with polymorphic form. Not only do the various crystal structures of a given pharmaceutical compound affect the efficacy of the drug, but they may also carry enormous legal implications. Much product revenue can depend upon the identification and patent protection of certain polymorphic forms. Thus, the control of crystallization is a very important process parameter, and techniques such as X-ray crystallography, infrared spectroscopy, Raman spectroscopy, and polarized light microscopy are routinely used in the characterization of crystalline drugs.

This presentation will involve the investigation of a variety of pharmaceutical polycrystalline films using infrared (IR) spectroscopic imaging. Preliminary data was collected using a conventional FT-IR microscope with visible polarized light capabilities. Correlating data was then collected using a commercially available IR imaging microscope.

In work reported previously we, and the McCreery Group at Ohio State University, have proposed the use of glass luminescence standards for the calibration of the Raman spectral intensity. We now have a program underway that is directed at the development of such secondary standards for the calibration of the Raman instrument response function. The first Raman intensity standard to be certified and issued by NIST this year will be for the relative intensity calibration of Raman spectra excited at 785 nm. in general, for any laser excitation wavelength, the calibration procedure is based on the use of luminescent glasses, doped with either transition metal or rare earth ions, that will furnish structureless, broad-band emission spectra upon laser excitation, over any chosen Raman range. For any selected glass standard, the intensity of the luminescence spectrum is calibrated versus the radiant output of a NIST-calibrated white light source, thereby transferring the white light calibration onto the output emission of the luminescent glass.

Single point near-infrared (NIR) spectroscopy is used extensively for characterizing raw materials and finished products in a wide variety of industries: polymers, paper, film, pharmaceuticals, paintings and coatings, food and beverages, agricultural products. As advanced industrial materials become more complex, their functionality is often determined by the spatial distribution of their discrete sample constituents. However, conventional single point NIR spectroscopy cannot adequately probe the interrelationship between the spatial distribution of sample components with the physical properties of the sample. to fully characterize these samples, it is necessary to probe simultaneously spatial and chemical heterogeneity and correlate these properties with sample characteristics.

Recently, we have developed a novel NIR imaging spectrometer that can deliver spatially resolved chemical information very rapidly. in contrast to conventional, single point NIR spectrometers, the imaging system uses an infrared focal-plane array (FPA) to collect up to 76,800 complete spectra, one for each pixel on the array, in approximately one minute.

Infectious diseases are the leading cause of death worldwide and the third leading cause in the US. Among these maladies are many that can be classified as emerging diseases. Some of these disorders may be caused by truly novel pathogens. in others, the causative organisms have been present for many years (for some, probably millennia), but have escaped detection until recently. Still others represent the re-emergence of known pathogenic organisms after a long period of quiescence. The mention of emerging pathogens brings to mind sensational exotic and feared microorganisms such as Ebola virus, human immunodeficiency virus (HIV), hantavirus, West Nile virus, Yersinia pestis (plague), and prion diseases such as bovine spongiform encephalitis (BSE, “mad cow” disease) which have been associated with variant Creutzfeldt- Jakob disease (CJD) in humans. However, other organisms that have been known for some time can be classified as emerging pathogens as they continually mutate, recombine, and adapt, causing misery and death.

Clinically, the combination of fever and hemorrhage can be caused by many pathogens, including viruses, rickettsiae, bacteria, protozoans, and fungi. However, the term viral hemorrhagic fever (VHF) is usually reserved for systemic infections characterized by fever and hemorrhage caused by a special group of viruses that belong to four different families: Filoviridae, Arenaviridae, Bunyaviridae, and Flaviviridae. All members are lipid-enveloped RNA viruses; except for filoviruses, where the resorvoir remains unknown, these viruses are transmitted to humans by arthropods and rodents. The VHFs are characterized by abnormal vascular regulation and damage. The VHFs all have similar clinical pictures with mortality rates of 15-30% or in the case of Ebola virus, up to 80%. Between 1993 and 2001, more different VHFs and related diseases were reported than during any comparable period over the past several decades. These diseases include Ebola VHF in Uganda in 2000, in Zaire in 1995 and in Gabon between 1994 and 1996; ongoing cases of Marburg hemorrhagic fever in the Democratic Republic of Congo beginning in 1998; Rift Valley fever in Saudi Arabia and Yemen in 2000 and in Kenya in 1997; Lassa fever in Sierra Leone in 1997; hantavirus pulmonary syndrome in the Americas since 1993; and imported cases of dengue hemorrhagic fever from Central and South America, and yellow fever from Africa during the 1990s.

Polyomaviruses are double-stranded DNA viruses of the family Papovaviridae. They replicate in cell nuclei, where they form 40-50-nm, non-enveloped, icosahedral nucleocapsids that exit the host cell by lysis (Fig. 1A-C). Polyomaviruses with pathogenic potential in both human and non-human hosts have been known for many decades. in recent years, however, new pathogenic polyomaviruses have been described in man, non-human primates, and other animals. New forms of acquired and iatrogenic immunocompromise have also led to changes in the prevalence and pattern of disease caused by well-characterized polyomaviruses. This presentation provides an overview of emerging polyomavirus diseases, illustrated with vignettes from our diagnostic practices. in many cases, the detection and monitoring of polyomavirus infections has been facilitated by ultrastructural examination.

Two well-characterized polyomaviruses cause disease in humans, almost invariably in the setting of immunosuppression. JC virus, described in 1971, is the causative agent for progressive multifocal leukoencephalopathy (PML), a generally fatal disorder characterized by patchy, progressive destruction of the cerebral white matter.

Sixteen years have passed since a microsporidian was first reported as an opportunistic pathogen in HIV/AIDS. to date, two new genera, Enterocytozoon and Trachipleistophora, and 5 new species, Enterocytozoon bieneusi, Encephalitozoon hellem and E. intestinalis, and T. hominis and T. anthropophthera have been described infecting patients with AIDS. E. bieneusi and E. intestinalis have been among the most common intestinal pathogens diagnosed as causes of diarrhea and wasting in HIV/AIDS.

Members of their phylum, Microspora, have in common a hollow polar tube that is tightly wound up in their spores. The coiling up of the polar tube, just beneath the cell wall, allows a structure that is several times its length to be packaged within a spore. E. bieneusi, which has the smallest of all microsporidian spores, 1 x 1.5 ¼m, overcomes its size limitations by packaging its polar tube in 2 layers of 3 coils each. When conditions are right, this unique structure is extruded from the spore and becomes the “needle of a syringe”.

During the 20th century, the advent of vaccines and antibiotics, and continued improvements in urban sanitation and water quality resulted in a dramatic decline in mortality due to infectious diseases. There was a brief period of optimism in the mid-1900's that infectious pathogens would no longer be a significant cause of death in the United States. For a number of reasons, this trend had reversed by the end of the century, and the rates of death caused by infectious diseases began rising. in recognition of this change, the Institute of Medicine issued a report in 1992 that called attention to the major factors contributing to the emergence and reemergence of infectious diseases. Among these were the following: increased human encroachment on wilderness habitats, resulting in changes in the ecosystem and increased contact with animal and insect vectors which may harbor unknown infectious agents; sizable growth in human population, resulting in increased urbanization and crowding; increased global travel; changes in human behaviors; and microbial evolution.

The basidiomycete fungus, Tilletia constitutes the most important group of smuts economically as they infect cereal grains which provide a major portion of the world’s food supply. The teliospores of the smuts which have been studied have a spore wall layer called the partition layer or the striated zone which is very resistant to fixatives and resins used for electron microscopy. We assume that the chemical nature of this wall layer is the primary factor to prevent spore desiccation and to maintain spore viability for many years. Teliospores of Tilletia tritici (Bjerk.) Wint. (formerly T. caries), T. controversa Kühn, T. indica Mitra, and Neovossia horrida (Tak.) Padwick and Khan all have a reticulated exterior spore layer (sheath). Common bunt, Tilletia laevis Kühn (formerly T. foetida) teliospores lack this exterior reticulated layer, but are also resistant to spore desiccation and maintain viability for many years. Therefore, the purpose of this study was to compare the ultrastructural characteristics of the partition layer of T. laevis teliospores with the partition layer of other teliospores of smut fungi which have been studied.

When it is all said, done, celebrated and published, the question remains, “How do we identify new viruses?” How do we build preparedness for rapid identification and teams whereby important data can be disseminated to our peers in virology, histopathology and molecular biology? The answers lie in the ability to build effective teams and to recognize the unique contributions of disciplines such as electron microscopy and the associated skill bases. The success in Australia for the rapid and accurate identification of new viruses has, in the main, been attributed to the effective use of electron microscopy. Data generated from such investigations have provided the foundation for subsequent studies involving the above areas of diagnostic inquiry. Data presented in this paper overviews some of the successes of electron microscopy and highlights its continued role in the identification of ‘emerging pathogens’ that have potentional impact on trade, human health, medical health and conservational biology.

The diagnosis of cytomegalovirus (CMV) in tissue sections is usually quite easy because of the characteristic appearance of intranuclear inclusions. At times the morphological features are equivocal, the nuclear findings may be atypical or only a single cell may be affected, thereby creating uncertainty about the diagnosis. This report reviews our experience with the various microscopic techniques that can be used to make definitive identification of CMV.

EM - Visualizing the viral particles in a single or even lysed cell by routine electron microscopy can make the definite diagnosis, but section size limits the usefulness in the detection of early CMV infection (fig. 1). However, if a diagnosis can not be made with certainty by the following methods, EM can be performed on stained paraffin sections for confirmation.

H&E - A Hematoxylin & Eosin stain that discloses characteristic intranuclear inclusions surrounded by a clear halo indicates CMV infection (fig.2A).

In the event of a bioterrorist attack, rapid screening, agent identification, and confirmatory diagnosis will be critical, so that prevention and treatment measures can be implemented quickly. However, because few biologic agents thought likely to be used as biological weapons represent major public health problems in the United States, we have limited capacity to diagnose them, either at the state and local or the federal level. to begin to address this new public health challenge the Laboratory Response Network for Bioterrorism (LRN) was established.

The LRN is a multi-level system designed to link state and local public health laboratories, clinical, military, veterinary, agricultural, water and food-testing laboratories. Operational in August of 1999, the LRN was established as a consequence of Presidential Decision Directive 39, increased congressional awareness of the huge biological weapons program in the former Soviet Union, the high level of risk to national security and subsequent emergency funding to the Centers for Disease Control and Prevention under the DHHS Operating Plan for Anti- Bioterrorism Initiative.

Twenty five years ago, in august 1976, a specialist workshop gathered in Cornell University, at John Silcox's invitation, scientists covering a broad spectrum of interests to assess the potential of analytical electron microscopy, as to instrumentation, fundamental limits, general level of data processing and current theoretical understanding. If the STEM instrument had already been existing for a few years in Crewe's laboratory, its major emphasis, in spite of the existence of an EELS spectrometer, had focused on new modes of high resolution imaging. At the first Cornell workshop, the STEM instrument was for the first time recognized as a potentially formidable analytical instrument because of the possibility of extracting all available signals simultaneously. Furthermore it was directly suitable for digital computer processing and therefore for providing quantitative information. It was also pointed out that a major advantage of the STEM would be its potential to record EELS spectra from every point in the field of view as one scans an area to form an image, thus offering the capability of "chemical" mapping beyond "elemental" mapping.

The typical electron micrograph is often rich in detail, sometimes too rich, and it can overwhelm the casual observer. The obvious interpretation may be quickly drawn and the next sample brought under study. Yet the electron interaction with the sample is rarely fully exploited. This may be the reason that electron microscopy may sometimes be dismissed as “just pretty pictures”. While in practice “pretty pictures” are not that easy to record or find, it is still true that the interaction of electrons with the sample is complex and therefore able to give the observer much more understanding than is normally found, provided only that the observer is able to unravel the observations. It is the rapid change in this situation that has been fascinating to watch unfurl during the past decades.

In the Scanning Transmission Electron Microscope (STEM) electrons are focused into a tiny spot that is scanned over a thin film specimen.