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.

STEM is an important tool for the study of material by forming Z-contrast ADF images. Studies of strain fields suggest that strain can cause extra contrast in ADF images. Since strain fields exist in many TEM samples, especially interfaces of two different materials, it is necessary to study the contrast effect of strain fields in more detail. in this paper, we study the contrast effect of strain fields by STEM experiments and multislice simulations.

Sample thickness and the dimensions of ADF detector can both affect strain contrast. We first study the influence of sample thickness. A cross-section TEM sample of an interface of crystal silicon and amorphous silicon was prepared by typical tripod polishing and was studied with the 100 kV Cornell VG 5.0 UHV STEM. The sample was tilted so that the crystal was on the (110) zone axis and ADF images of the interface were recorded.

Annular Dark Field imaging has the potential to be directly quantifiable. By this I mean that with careful measurement, the absolute image intensity has physical meaning. Unlike Bright Field TEM, the ADF image has no contrast reversals with focus and with the exception of thick specimens there are no contrast reversals with changes in thickness. Thus, image intensity is related to thickness, composition, orientation, and structure of local regions whose size is determined by the electron probe. The ability to extract quantitative information about the specimen from the intensity requires careful collection of the intensity data and a solid understanding of how that intensity will change with thickness, composition, orientation, and structure. The qualitative effect of thickness and composition has been well shown in the literature but more quantitative approaches have been lacking.

The simplest models of ADF imaging treat each atom interacting

Many of the topics in valence spectroscopy originally pioneered with TEM equipment have now been taken over by STEM. Notable exceptions are the studies by J. Silcox and his colleagues of plasmon dispersion and of the guided modes in thin slabs where retardation and other relativistic effects arise. The growing use of STEM valence EELS on increasingly complex nanostructures and the development of dielectric excitation theory for quantitative interpretation of the results were recently reviewed. The spatial resolution approaches the atomic scale, exceeding by orders of magnitude the capability of near field spectroscopy. By using the cathodoluminescence (CDL) signal, optical precision in STEM energy loss measurement can also be achieved, particularly in the difficult region below 5 eV or even 1 eV. High spatial resolution can be preserved provided that an intermediate stage of carrier diffusion is not involved in the radiative decay of excitations.

Figure 1 shows STEM CDL images of an Ag particle obtained from the dipole l=1 and quadrupole l=2 surface plasmon excitations

Carbon nanotubes (CNT) have great potential as one-dimensional quantum conductors in future molecular electronic applications. Local electronic property determinations at low energies in CNT have been confined to those made by scanning probe microscopy techniques (SPM). Although SPM measurements provide unprecedented electronic property information, they suffer from substrate coupling and a limited ability to measure structural characteristics of nanotubes. A high-resolution transmission electron microscope (TEM) coupled with an electron energy loss spectrometer (EELS) offers electronic property determination as well as structural (imaging and diffraction) and spectroscopic analysis of truly isolated nanotubes. Here we report measurements of EEL spectra of single-walled CNT, both singly and in bundles. The experiments were performed in a VG HB501 STEM at Cornell University, operating at 100 kV. CNT samples chosen for analysis include isolated single-walled tubes with diameters 1.2-1.3 nm and bundles of ∼3-150 single-walled tubes. Many of the spectra were obtained in the aloof mode, i. e. with the beam not penetrating the material.

There is an intimate connection between the electronic structure of a material and its physical properties. to change one, is to change the other. Some of the most striking illustrations of this relationship can be found at grain boundaries in metals and their alloys. Here, the most important changes in cohesion can be described by changes in the local density of states (LDOS), which in turn can be measured using EELS [1]. The first demonstration that EELS could be used to connect the electronic and mechanical properties of a material was in revealing the role that boron has in restoring a bulk-like bonding to grain boundaries in Ni3Al [2,3]. Boron was known to change the fracture mode in Ni3Al from intergranular to transgranular, possibly by enhancing grain boundary cohesion.

What interested me in this project, when John Silcox first suggested it as a thesis topic, was the potential of using the EELS fine structure to measure materials properties directly.

Magnetic tunnel junctions (MTJ) which consist of two ferromagnetic electrodes separated by a thin (∼10Å) tunnel barrier can exhibit a magnetoresistance of greater than 40% at 295 K. They hold substantial potential for application as computer memory and magnetic field sensors. Much about the spin-dependent tunneling process is still not understood. in order to identify features that may affect the spin-polarization of the tunnel current, we have used the UHV STEM at Cornell University to perform atomic-scale imaging and EELS on an MTJ system. The MTJ was sputter deposited on (100) Si with layer sequence Co 60Å/Al 12AÅO2 exposure/Co 100Å.

The annular dark field (ADF) image (Fig. la) shows the A10x barrier layer has a fairly uniform thickness of about 15 Å. Its profile (Fig. 1c) appears diffuse partly due to layer roughness rather than atomic mixing. in bright field (BF) the appearance of strong Fresnel fringes (Figs. 1b,c), due to a large difference in average electrostatic potential between Co and AlOx, complicates interpretation.

For failure analysis and process control it is critical to perform comprehensive microanalysis. The desire to perform this analysis thoroughly but quickly is the ultimate goal. Typical analyses of this sort have been performed by collecting x-ray spectra at points, along lines, by mapping, and more recently by spectrum imaging where a complete x-ray spectrum is collected over a 2D array of points. Spectrum imaging is perhaps of greatest interest for its potential to thoroughly and comprehensively sample a given area of a microstructure. The problem is that the techniques available for analyzing large (> 1000 spectra) spectrum image (SI) data sets have been limited to mapping after the fact and perhaps thresholding the maps and summing the resultant spectra from those pixels. As a result, there is a need for more sophisticated SI analysis tools, capable of reducing the SI to its essence-the chemical components. Software is currently commercially available for the automated analysis of x-ray spectrum images (SIs) from the SEM

Energy Dispersive Spectrometry (EDS) is an ubiquitous method of elemental analysis for SEM, TEM, and STEM applications. The elements of interest are generally quantified without standards using theoretical calculations or by using standards that are high purity specimens of the elements measured. However, EDS is often used to determine a small percentage of an element in a matrix. The accuracy and limit of detection of these low concentration measurements has not been established. An earlier report proved the concept that a cross section high dose BF2 implanted specimen could provide a standard for EDS measurement of F. This study extends this quantification approach to transition elements of importance to the semiconductor industry.

The Fe and Co standards were created by high dose ion implantation. For ions implanted into silicon, a dose of lxl016 atoms/cm2 results in a peak concentration of approximately lxl021 atoms/cm3 or 2% atomic. The exact concentration can be determined using methods such as Rutherford Backscattering Spectrometry (RBS) and Secondary Ion Mass Spectrometry (SIMS).

CdSe/ZnSe based semiconductor quantum dot (Q D) structures are a promising candidate for optoelectronic device applications. However, key to the luminescence properties is the cation distribution and ordering on the atomic level within the CdSe QDs/agglomerates. Here the Z contrast imaging technique in the scanning transmission electron microscope (STEM) is employed to study multisheet (Cd,Zn,Mn)Se QD structures. Since Z-contrast is an incoherent imaging technique, problems associated with strain contrast in conventional TEM are avoided an accurate size and composition determinations can be made.

For this work we used a JEOL JEM 201 OF field emission STEM/TEM. The sample was grown by molecular beam epitaxy in order to achieve vertical self-ordering of Cd rich quasi-2D platelet This sample comprises 8 sequences of 10 ML (2.83 nm)Zn0.9Mn0.1Se cladding layer and 0.3 ML (0.09 nm) CdSe sheet, a further 10 ML of Zn0.9Mn0.1Se, and a 50 nm ZnSe capping layer.