Recent developments promise the possibility to externally control every aspect of microscopes through a computer interface. In combination with high-resolution cameras and feedback to the microscope, this can be leveraged to create highly automatic routines, e.g., to remotely correct astigmatism. Together with the development of fast computer networks this creates a new branch of microscopy, the so-called “telemicroscopy”. The goal of telemicroscopy is the control of a microscope over a large distance including the transfer of images with an acceptable repetition rate. A big advantage for electron microscopy in particular is the possibility of having access to expensive and well-equipped microscopes. In the field of light microscopy the branch “telemedicine” was created, meaning the “virtual” presence of a colleague or specialist for discussion or diagnosis.

Using transmission electron microscopy as an example, the history and special requirements for automation and telemicroscopy will be discussed. In the late 80's the first TEM with a remote control was revealed. Shortly thereafter, first automatic functions for defocus control and astigmatism correction were developed using a video camera as electronic image converter.

This issue of Microscopy and Microanalysis contains a selection of invited papers from the topical symposium, Thirty Years of Energy-Dispersive Spectrometry in Microanalysis, sponsored by the Microbeam Analysis Society (MAS) at the Microscopy & Microanalysis (M&M) '98 meeting, held July 12-16 in Atlanta, Georgia. This was the second MAS topical symposium held in conjunction with the annual M&M meetings, the first being at the meeting in Minneapolis, Minnesota, in 1996. The MAS topical symposia are part of an initiative to provide venues for in-depth study of topics of special interest to the microbeam analysis community. The next MAS topical symposium is planned as part of M&M '99 and will celebrate Fifty Years of the Electron Microprobe.

Cryo-high-resolution scanning electron microscopy was used to analyze the conformation of fibronectin fibrils formed in human skin fibroblast cultures or in a cell-free system by treating soluble plasma fibronectin with guanidine. Structurally, fibrils assembled in the cell-free system and in culture were similar. Assembly of both fibrillar networks involves interactions with the III1 and amino terminal repeats of fibronectin; their conformations consist of either smooth surfaces or patches of smooth surfaces and nodules randomly spaced along the fibril. The random distribution of these two conformations in fibrils indicates that fibronectin fibrils are capable of undergoing localized conformational changes. The nodules may be discrete domains of 3 to 4 type III repeats, as they can be labeled with the monoclonal antibody IST-2 to the III13-14 repeats in fibronectin and are found in 160 kDa and 85 kDa fragments of fibronectin that only contain type III repeats. In our study, smooth regions of fibrils were never recognized by the IST-2 antibody, suggesting that the epitope in the III13-14 repeats is masked in these regions. These results demonstrate that fibronectin fibrils are flexible and certain epitopes of fibronectin may be buried, or exposed, depending on the conformation of the fibril. They also show that fibrils assembled in cell-free conditions can be a powerful tool for studying fibril formation.

Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of vertebrates as well as nonpathogenic organisms living in symbiotic association with their tick hosts. We investigated the ability of SFG rickettsiae to move between and within host cells using tick cell culture and a SFG rickettsial isolate from a lone star tick (Amblyomma americanum) collected in Missouri. The isolate (MOAa), which is closely related to Rickettsia montana, grew in cell lines from the ticks Ixodes scapularis and Rhipicephalus appendiculatus. Transmission electron microscopy demonstrated that immediately following entry into tick cells, rickettsiae escaped from the host cell membrane, and intracellular rickettsiae came to lie in direct contact with host-cell cytoplasm. There was evidence of damage to the endoplasmic reticulum membrane which was broken down into vesicular structures. When rickettsiae exited host cells, host membrane stretched around them but was lost before re-entry. Use of a fluorescein-tagged monoclonal antibody to rickettsial outer membrane protein B and rhodamine-labeled phalloidin demonstrated association of actin tails with rickettsiae and suggested that SFG rickettsiae utilized host cytoskeletal components for movement. During early stages of infection, when cells harbored only one or a few organisms, “comet tails” of F-actin formed on one end of rickettsial cells, presumably pushing them ahead. Actin tails were not seen during later stages of infection when tick cells became completely filled with rickettsiae.

Dear Colleagues: Please take a moment to read the new Aims and Scope statement found on page 188. This statement has recently been revised in response to comments indicating that the previous statement could be interpreted as not encouraging submission of manuscripts that describe mainly applications, particularly in the biological fields. That is clearly not the case, and the Aims and Scope statement has been revised in this issue to correct that misperception.

A Monte Carlo model was used to simulate specimen-electron beam interactions relevant to electron back-scattered diffraction (EBSD). Electron trajectories were calculated for a variety of likely experimental conditions to examine the interaction volume of the incident electrons as well as that of the subset of incident electrons that emerge from the specimen, i.e., back-scattered electrons (BSEs). The spatial resolution of EBSD was investigated as functions of both materials properties, such as atomic number, atomic weight, and density, and experimental parameters, such as specimen thickness, tilt, and incident beam accelerating voltage. These simulations reveal that the achievable spatial resolution in EBSD is determined by these intrinsic and extrinsic parameters.

In the microelectronic semiconductor world we are bombarded with reports of how the drive toward faster, denser, lower power-consuming and more reliable semiconductor products will accelerate with time. This paper discusses the instrumental evolution from visible light microscopy to scanning electron microscopy and on to transmission electron microscopy and scanned probe microscopy. The increased demands placed on specimen preparation of precise locations in a semiconductor chip for microscopy are discussed. Analytical microscopy has to be timely in order to be a viable adjunct to semiconductor manufacturing. The factors governing analysis of turn-around time are explained and an optimum strategy is suggested for effective utilization of finite laboratory resources. The new instrumentation available to the microscopist is introduced along with an overview of the exciting new analyses that will be available in the immediate future.

Electron beam damage is a significant limitation for transmission electron microscopy (TEM) studies of beam-sensitive samples. An approach for studying surface reactions on alkali halide crystals using 200 kV TEM is presented. Experiments were designed to monitor the reaction of NaCl crystals with HNO3 gas followed by water vapor to form solid NaNO3. During beam damage experiments, TEM micrographs record structural changes to both NaCl and NaNO3, including dislocation loops, void formation, and decomposition. Sample decomposition can be successfully minimized by a combination of commonly used techniques: (1) focusing the beam adjacent to the area of interest, (2) lowering the electron density, (3) choosing to image larger (micrometer- versus submicrometer-sized) alkali halide crystals, and (4) lowering temperature by the use of a liquid nitrogen cooling stage. From these results, additional studies were designed that monitored sequential experiments. Sensitive micrometer-sized sodium chloride single crystals before and after exposure to nitric acid vapor and water vapor and the subsequent growth of submicrometer-sized sodium nitrate single crystals could then be successfully imaged using TEM.

This issue of Microscopy and Microanalysis contains invited and contributed papers from the 1998 Arizona State University (ASU) Workshop on In Situ Electron Microscopy, held January 7-10 at the Embassy Suites in Scottsdale, Arizona. This was the eighteenth in a series of Workshops organized annually by the Facility for High Resolution Electron Microscopy in the Center for Solid State Science at ASU. The Workshops are part of an educational program, and are intended to foster interaction and collaboration between researchers in electron microscopy. The workshop was attended by over 80 participants from universities, industries, and government laboratories around the world. Many of the contributions to the workshop are included in this issue.

The examination of archival pathology specimens of human small intestine by light microscopy, field emission scanning electron microscopy (FESEM), and confocal scanning laser microscopy using fluorescent in situ hybridization (FISH) techniques was undertaken to better understand the epidemiology of Giardia. Giardia trophozoites were tentatively identified in the light microscope after hematoxylin and eosin (H&E) staining. The organisms were adherent to the intestinal epithelium where they were also associated with strands of mucus within the lumen. Fluorochrome-conjugated antisense oligonucleotide probes, developed for the 16S rRNA of Giardia lamblia and Giardia muris, were used in FISH experiments with confocal scanning laser microscopy. Positive identification of trophozoites could be obtained with the G. lamblia probe, but not with the G. muris probe. FESEM examination of serial sections adjacent to FISH-stained sections revealed trophozoites characterized by their morphological features. The 16S rDNA probes specifically distinguished different species of Giardia, but whether multiple infections can occur within an individual host could not be determined.

Specimens that charge under electron beam irradiation in the scanning electron microscope (SEM) can be stabilized by choosing the beam energy to be such a value that the sum of the secondary and backscatter electron yields is unity, as this establishes a dynamic charge balance. We show here that for pure elements, the energies El and E2, for which charge balance occurs, are related directly to the atomic number of the material. Although generally there is no comparable relation for compounds, we also show that for polymers, the E2 energy is related both to the ratio of the number of valence electrons to molecular weight and to the electro-negativity of the monomer units that form the polymer.

The field of in-situ electron microscopy has recently become very active. Microscopy and Microanalysis 1997 was host to an extended symposium devoted entirely to in-situ studies. Also, a recent Winter School sponsored by Arizona State University was devoted entirely to in-situ electron microscopy and drew over 90 attendees. Examination of the presentations and papers in these in-situ symposia has revealed that the researchers involved still have problems acquiring, managing and storing and manipulating their data. An in-situ experiment, by its very nature, is dynamic and recording micrographs is typically not the optimum method of image acquisition. Videotape is the preferred medium. High grade S-video recording of experiments permits the extraction of single frames of reasonable quality from the tapes for presentation and publication. There are, however, a number of problems with video recording. During in-situ studies a number of variables in the sample environment are changing, e.g. the pressure, the temperature, the stress, the strain and the microscope parameters (accelerating voltage, beam current, magnification, specimen tilt, etc.).

Particle size distributions obtained from a thin section are usually a skewed version of the true distribution. A previous method for determining the parent distribution was questionable because negative particle frequencies could be obtained. Here, we describe a method of determining parent distributions of spherical particles using a model with adjustable parameters. Our calculated distributions are somewhat broader than the distributions obtained with previous methods, but the average particle sizes are nearly identical. The newly developed model is applicable to any type of transmission microscopy.

The energy-dispersive Si(Li) X-ray spectrometer, introduced 30 years ago into electron probe mi-croanalysis (EPMA) by R. Fitzgerald et al., has profoundly affected the development of microanalysis. It offers many advantages over the wavelength-dispersive crystal spectrometer. It has no moving parts and covers the full energy range of interest in EPMA. There is no defocusing over large distances on the specimen, the efficiency of the device is high, varies slowly and continuously with atomic number, and can be predicted fairly accurately, and, most importantly, all emission lines are detected and can be observed simultaneously. The one remaining disadvantage of the Si(Li) spectrometer is its poorer energy resolution. Solid-state detection devices now under development promise to achieve resolution comparable to that of the crystal spectrometer.

A focused ion beam (FIB) interface attached to a column of 200 keV transmission electron microscope (TEM) was developed for in situ micropatterning to semiconductors. TEM specimens of Si and GaAs, and those of a thin Ni2Si layer on a Si substrate were micromilled in the TEM during observation. A set of 6 x 6-um squares and alphabet letters were patterned with a 25 keV Ga+-FIB of 0.2-μm beam diameter at room temperature. The effect of FIB irradiation on the structural evolution was observed simultaneously by a TV-rate video camera and sequentially by regular film. FIB micropatterning to semiconductor specimens caused amorphization and Ga injection. The excess Ga in the specimens precipitated as metastable solid γ-phase for Si and as liquid phase for GaAs. Ni2Si/Si specimens lost silicide crystallinity after FIB patterning. Annealing of these bilayer specimens at 673K resulted in the precipitation of Ni-rich silicide.

As part of the Microbeam Analysis Society (MAS) symposium marking 30 years of energy-dispersive spectrometry (EDS), this article reviews many innovations in the field over those years. Innovations that added a capability previously not available to the microanalyst are chosen for further description. Included are innovations in both X-ray microanalysis and digital imaging using the EDS analyzer.

Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma-mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.