Sea urchin embryos have served as a model system for the investigation of many concepts in developmental biology. Their gastrulation consists of two stages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proliferation of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate rigorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, we developed a new method for cell tracking based on two-photon excited photorelease of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cells throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biological damage. Cellular involution into the archenteron was observed throughout primary and secondary invagination, and the larval intestine was formed by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Further, as two-photon excitation microscopy becomes accessible to many researchers, the novel techniques described here will be broadly applicable to development of other invertebrate and vertebrate embryos.

The performance characteristics of electron microscopes and probe-forming instruments depend ultimately on the focal properties and aberrations of electron lenses. A practical method of experimentally determining the properties of electron lenses is described. The method utilizes shadows cast by two meshes inserted separately in front of the lens and behind the lens to study the properties of the image of a point source. The image properties are then used to calculate the lens properties. The paraxial values of the focal length and focal distance as well as their spherical and chromatic aberrations are determined. Experimental data and the analysis are presented in the form of a tutorial that has been tested in the classroom. Discussions of the relationship between image properties and lens properties, in particular, focal point aberrations and focal length aberrations, and the various ways aberration coefficients can be defined, are included to clarify concepts in optics that are important for microscopists.

A method for preparing a cross-section sample from a free-standing thin-film coupon suitable for transmission electron microscopy analysis has been successfully developed. The free-standing coupon presented a significant challenge for the preparation of a cross-section sample. A stack consisting of a silicon wafer, thin-film coupon, and glass coverslip was epoxied together, providing both support for thin film and enough surface area to handle the specimen during the sample preparation procedure. The tripod polisher wedge technique was used to thin the cross-section sample to 2 to 5 μm in thickness. This produced a sample with the required structural integrity. Final thinning was accomplished using an ion mill. This technique showed no artifacts introduced from the sample preparation procedure.

The Materials Microcharacterization Collaboratory (MMC) was created last year as a pilot project within the US Department of Energy's DOE2000 program. The DOE2000 program has three main goals: improved ability to solve DOE's complex scientific problems, increased R&D productivity and efficiency, and enhanced access to DOE resources by R&D partners. One of the strategies to meet these goals is the construction of national collaboratories. These provide integration on the Internet of unique or expensive DOE research facilities and expertise for remote collaboration, experimentation, production, software development, modeling, or measurement. In addition, collaboratories will benefit researchers by providing tools for video conferencing, shared data-viewing, and collaborative analysis. Cooperative pilots projects, jointly funded by DOE2000 and a scientific program area, are expected to lead to clear success in the scientific environment: they are to provide new capabilities and to increase the efficiency of doing the work (e.g., reduce travel, increase communication, provide for sharing of data among formerly disparate environments).

X-ray microanalysis of materials using energy-dispersive spectrometry (EDS) has made the greatest impact in studies of compositional changes at atomic-level interfaces. The small physical dimensions of the silicon detector make EDS the X-ray analyzer of choice for analytical transmission electron microscopy (AEM). X-ray analysis of thin foils in the AEM has contributed to our understanding of elemental segregation to interphase interfaces and grain boundaries, as well as other planar defects. Measurement of atomic diffusion on a small scale close to interphase interfaces has permitted determination of substitutional atomic diffusivities several orders of magnitude smaller than previously possible and has also led to the determination of low-temperature equilibrium phase diagrams through the measurement of local interface compositions. Elemental segregation to grain boundaries is responsible for such deleterious behavior as temper embrittlement, stress-corrosion cracking, and other forms of intergranular failure. On the other hand, segregation can bring about improvement in behavior: sintering aids in ceramics and de-embrittlement of intermetallics. EDS in the AEM has been responsible for quantitative analysis of all aspects of the segregation process and, more recently, in combination with electron energy-loss spectrometry (EELS) has given insight into why boundary segregation results in such significant macroscopic changes in properties.

Light microscopy was used to analyze the effects of added catalyst at different conditions (temperature and reaction times) in liquefaction testing of a low pyritic sulfur bituminous coal. Quantitative changes in vitrinite/vitroplast reflectance of coal and liquefaction residues were shown to be useful markers in analyzing and understanding the role of catalyst during the initial stage of coal particle hydrogenation. Lower reflectance values corresponded to increased conversions up to about the 60 min and 375°C experimental conditions. Microscopical observation of liquefaction residues also revealed the presence of “wall scales” of varying width.

Time-resolved high-resolution transmission electron microscopy at a spatial resolution of 0.2 nm and a time resolution of 1/60 sec using a piezo-driving specimen holder is reported here. Various types of atomic processes in mechanical interaction, such as contact, bonding, deformation, and fracture, in nanometer-sized gold crystallites and carbon nanotubes are demonstrated.

The quantity and spatial distribution of calcium bound to myofilaments is of considerable interest to physiologists studying the regulation of contraction in striated muscle, but few techniques allow these distributions to be directly measured. Methods are described here by which such data can be derived from electron probe X-ray microanalysis (EPXMA) images. Combination of data from multiple half sarcomeres improves the signal-to-noise level and allows assessment of variation between half sarcomeres. Profiles of calcium counts, calcium counts normalized to local bremstrahlung counts, and calcium counts normalized to the average bremstrahlung counts for the entire half sarcomere provide differing but complementary information. Such data help to identify and characterize spatial variations in Ca binding to regulatory proteins in muscle.

In this report, we describe the implementation of an angle-gating mechanism into a reflection optical microscope for imaging through a turbid medium. A significant improvement in image resolution and contrast is obtained when a polarizing annular objective is used to image a reflective test bar through various turbid media. The experimental results confirm that the improvement is caused by the efficient suppression of diffused photons scattered from the turbid medium when a polarized annular objective is employed as an angular gate.

We are developing a testbed for automated identification of specimens, and acquisition of high resolution images for 3D macromolecular EM. Several software package which have been developed specifically for acquisition of tomographic data sets have illustrated the benefits of automation. These software packages require the user to identify a specimen of interest and then automatically acquire a single tilt series from the identified specimen. The testbed we are developing will provide a generalized system for specimen identification, image acquisition and image quality assessment. The goals of the testbed are to provide a system that will acquire EM images unattended by an operator.

The testbed integrates a computer controlled TEM with a distributed real-time image processing environment. Computerized control and image acquisition from the TEM uses the emScope software package (1). This is coupled to existing image processing software packages and new software tools developed specifically for this project.

We have developed a microwave protocol for a paraffin-embedding microtechnique of the shoot apical meristem of Zea mays and have successfully applied this protocol to other plant tissues. This protocol decreases the time required for all aspects of microtechnique tissue processing, including fixation (24 hr to 15 min), dehydration (73 hr to 10 min), and infiltration (96 hr to 3 hr). Additionally, the time required to adhere paraffin ribbons to gelatin-coated slides and for the Johanson's safranin O, fast green FCF staining protocol has been significantly decreased. Using this technique, the quality of tissue preservation and subsequent in situ localization of knotted mRNA was increased by using microwaves.

The kinetics of a thin-film, solid-state reaction were investigated in the spinel-forming oxide system Fe2O3/MgO. In this study, epitactic thin films of Fe2O3 (α, or corundum, structure) were deposited on (001)-oriented MgO using pulsed-laser deposition (PLD). The resulting diffusion couples were then reacted at elevated temperatures in air to induce the reaction between the thin-film and bulk substrate to form the spinel, MgFe2O4. Both the as-deposited and reacted diffusion couples were characterized using low-voltage scanning and transmission electron microscopy. These techniques allow the kinetics of the reaction and the structural properties of the spinel to be investigated.

A laboratory test of X-ray tomography employing a diverging beam of X-rays rather than the usual parallel X-ray beam is described. We chose to test and demonstrate the advantages of divergent beam tomography by imaging an extracted juvenile human premolar using an ordinary dental X-ray source and a cooled CCD camera. Experiments with a three-piece cover-glass sample and with the human tooth demonstrated that three-dimensional reconstruction can be achieved at 34 μm per pixel resolution employing an X-ray tube spot 800 μm in its smallest direction without requiring close contact with the fluorescent screen. Reconstruction of a 256 x 256 pixel single-plane image from 100 projection images took only 45 sec on a personal computer with a Pentium 166 MHz processor. We have also demonstrated a volume reconstruction of 256 x 256 x 256 voxels from the data. Successful extension of this work to submicrometer projection X-ray microscopy is predicted. Improved resolution of medical tomography is another possible application.

Three-dimensional (3D) grain sizes and size distribution functions (SDFs) are generally obtained by converting the 2D or ID grain sizes to 3D with the methods based on a spherical grain shape model (S-model), which can produce large systematic errors. In this report, a new polyhedral grain model (P-model) is developed and experimentally checked with the directly measured 3D grain sizes. The results show that the systematic error originating from the S-model can be corrected by using the new polyhedral grain model.

The Microscope and Graphic Imaging Center (MAGIC) at California State University, Hayward, has developed a model for interactive remote shared access to visual and analytic scientific instrumentation (IRSA/VASI). Using specialized software over the Internet, instruments such as electron microscopes can be shared throughout the world. Remote access to a centralized microscope provides investigators with specialized equipment that they might not be able to purchase and support on their own. For those who already have such instruments, it affords access to additional equipment and permits them to use their own resources in a wider context. Remote control of instrumentation is being researched and developed at national laboratories, supercomputing centers, and universities. Remote access and control was successfully demonstrated in 19921 and since that time, there have been other major achievements.

To demonstrate our model of interactive remote shared access to instrumentation, we have developed a system of software programs for remote control of a Philips XL-40 scanning electron microscope.

Design rules are described here for high-temperature straining stages for transmission electron microscopy. Temperatures above 1000°C can be attained by electron bombardment of the specimen grips. Thermal equilibrium can be reached in a short time by carrying off the heat by water cooling. Some applications of this stage are described. Ferroelastic deformation was observed at 1150°C in t′ and partially stabilized zirconia, which changes the microstructure for successive dislocation plasticity. In the oxide-dispersion-strengthened alloy INCOLOY MA 956, dislocations are impeded by oxide particles and move smoothly between the particles. At high temperatures, both the resting and traveling times control the average dislocation velocity. In MoSi2 single crystals of a soft orientation, dislocations with 1/2〈111〉 Burgers vectors are created in localized sources and move on {110} planes in a viscous manner. The dislocations in Al-Pd-Mn single quasicrystals are oriented in preferred crystallographic directions and move in a viscous way as well. On the basis of in situ observations, conclusions are drawn for interpreting macroscopic deformation behavior at high temperatures.

Low-voltage energy-dispersive X-ray microanalysis has a number of distinct advantages for measuring the concentration of light elements (Z = 11−20) in a largely organic matrix. Between 10 and 4 kV, there is a 50-fold decrease in the size of the incident beam-specimen interaction volume which enables discrete subcel-lular compartments to be analyzed. Experiments with mineral and organic samples of known chemical composition show that two of the most widely used analytical algorithms are capable of providing quantitative data at accelerating voltages as low as 5 kV. This technique is used at 5 kV to analyze frozen hydrated fracture faces of leaves of the tea plant which are known to contain aluminium. Higher levels of aluminium are found in the cell walls and are associated with increased levels of silicon and magnesium. The advantages and disadvantages of the experimental approach are discussed in relation to analysis carried out at higher voltages.

A technique has been developed to provide real-time imaging, with several nanometer resolution, of organometallic chemical vapor deposition (CVD) by scanning electron microscopy under conditions approaching those used in the microelectronics industry. The technique involves modifications to an environmental scanning electron microscope (ESEM) to facilitate organometallic precursor gas handling and sample heating. To demonstrate the usefulness of this technique for studying the microstructural evolution of CVD-grown metal films, results of Al/SiO2 CVD experiments are presented.