The cAMP dependent-protein kinase (PKA) signaling pathway plays a key role in the sympathetic regulation of muscle contraction in adult cardiac myocytes. This pathway may be regulated via compartmentalization of its components, whereby A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular areas to give each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid, and the regulatory subunit of PKA (R).

Fluorescence resonance energy transfer (FRET) was used to monitor binding events between the type II regulatory subunit of PKA (RE) and Ht31 or Ht31P, a mutated form of Ht31 which does not bind RII. Each protein was fused to a derivative of the green fluorescent protein (GFP) so that the excitation-emission spectra of the two fluorescent proteins overlap.

The widespread availability of automated fluorescence microscope systems has led to an explosion in the acquisition of digital images by biologists. This has created a need for computer applications that automate the analysis of these images and an opportunity to develop new approaches to classical problems. An example is the determination of the subcellular location of a protein from immunofluorescence images (or, more recently, images of GFP fluorescence). Current practice is to compare such images to mental images that a cell biologist has developed over time, and to reach a tentative conclusion about the structure (i.e., organelle) that a protein is found in. Since this determination is subjective, it often must be followed up by double labeling with a marker protein from the suspected structure.

As an initial exploration of the feasibility of automating the determination of subcellular location, we developed a system that is able to classify the localization patterns characteristic of five cellular molecules (proteins and DNA) in Chinese Hamster Ovary (CHO) cells. Images were acquired on an epifluorescence microscope after the cells had been fixed, permeabilized, and labeled with appropriate fluorescent reagents (usually antibodies conjugated to fluorescent dyes). The labels used were directed against a Golgi protein, a lysosomal protein, a nuclear protein, a cytoskeletal protein, and DNA.

A partnership between California State University, Hayward and Ohlone College, Fremont, California resulted in a program to introduce undergraduate students to scanning electron microscopy (SEM) sample preparation procedures. Ten students enrolled in a biology major's course at the Ohlone Community College are selected each semester to participate in the program. In addition to these students, ten others from a Basic Skills Enhancement program for underrepresented students participate through a short course offered during the summer. The program, in effect for two years, resulted in the compilation of a laboratory manual describing basic sample preparation techniques that are appropriate for an outreach program. We select samples that are simple to prepare yet yield results that are germane to a biology student's curriculum. A selection of plants, pollen grains, bacteria, fungi, yeast, Drosophilia melanogaster, and various other invertebrates are typical samples that lend themselves to an outreach program that emphasizes biological concepts. Initially our program required the Ohlone College students to come to the Hayward campus to prepare their samples. External funding has allowed us to purchase sample preparation equipment for the community college.

Bugscope is a new educational project in the World Wide Laboratory. The World Wide Laboratory provides Web browser based control of scientific imaging instrumentation using the Internet [1]. Providing K-12 classrooms with web based remote access to sophisticated scientific imaging systems was initially demonstrated by us in 1996 in the Chickscope project [2,3]. Chickscope allowed students to study chicken embryo development using a remotely controlled magnetic resonance imaging (MRI) system from their classrooms. While the Chickscope project was highly successful, the resources required to provide operational support for the remote imaging aspects of the project for a small number of classrooms were enormous and the project was not sustainable. The Bugscope project builds on the methods developed and the lessons learned from the Chickscope project. The primary goal is to demonstrate that relatively low cost, sustainable access to an environmental scanning electron microscope (ESEM) can be made available to K-12 classrooms to examine arthropods.

Methods: Classrooms use a standard web browser over the Internet to control and acquire images from a Philips XL-30FEG ESEM. The architecture to support remote acquisition is shown in fig. 1. The client/server control architecture for the ESEM remote control server is based on the emScope control library [4].

The focused ion beam (FIB) instrument has been developed and exploited by the microelectronics arena for specimen preparation for both scanning and transmission electron microscopy (TEM). The inception [1] and subsequent development [2] of the FIB TEM lift-out (LO) technique has enabled electron transparent membranes of generally uniform thickness to be produced for TEM analysis. The primary advantage of the FIB technique is that site specific cross sections (or plan view sections [3]) may be fabricated quickly and reproducibly. The FIB LO technique has been used extensively in our laboratory for a wide range of materials [4] and biological applications [5] which are summarized in figure 1.

The FIB LO method consists of milling a series of trenches around an area of interest. Then the bulk sample is tilted up to ∼60 degrees to allow the beam to impinge on the lower portion of the specimen surface so that cuts can be made along the bottom edge and the lower 2/3 of the distance up one side of the specimen.

X-ray mapping is one of the most popular modes for displaying information obtained with x-ray spectrometry performed in the scanning electron microscope. This popularity arises from the ready accessibility and apparent simplicity of information presented in a pictorial fashion, especially when used in conjunction with other SEM imaging modes, such as backscattered, secondary, and specimen current electron images. Further, the rise of powerful, inexpensive computer systems capable of image processing and display has given the analyst a dedicated, on-line tool with the capacity and flexibility needed for problem solving. Figure 1 shows a typical example of mapping. Although the interpretation of x-ray images obtained with a modern digital control and recording system would seem to be straightforward and relatively trivial, there are significant pitfalls and limitations that can easily fool the unwary. In Figure 1, within an individual x-ray map, the observer can reasonably judge where the concentration is lower or higher, at least for a group of contiguous pixels. Can such judgments be made among a set of maps of the same region for different elements, or even for the same element from different regions of the same specimen? With current x-ray processing and display systems, the answers are generally no. In fact, problems that can influence interpretation can arise at each stage of x-ray generation/emission, x-ray spectral collection, processing, and display.

Since the introduction of personal computers in the early 1980s, most documents and manuscripts have been prepared from within a word processing program on such a computer. Indeed, most documents on personal computers are created on a limited number of word processing packages that have come to dominate the market. Most of the data acquisition that attendees of Microscopy and Microanalysis currently perform is digital, i.e. directly into a personal computer or desktop workstation. It is frequently the case that images, spectra and diffraction patterns are only committed to paper when they are dispatched for publication. With the advent of the World Wide Web in 1994, and its subsequent explosive growth, entirely electronic publishing has become possible. It is possible to envisage the day when most publications will be available solely electronically and material will be only printed out occasionally by the reader.

While there are a growing number of journals that are only available electronically, entirely paperless publication is not yet the norm and there are no definite plans, as yet, to publish the proceedings of the Microscopy and Microanalysis meetings solely in electronic format. However, there are a number of advantages to applying some of these emerging technologies in the production of the future proceedings. Electronic submission of abstracts is one advance that is being considered by the organizers of the meetings scheduled for the early 2000s. The National Science Foundation now receives almost all proposals electronically. There are several reasons for moving to electronic submission. The first is that proofing and correcting the abstracts would then be straightforward.

There are several forms of three-dimensional (3-D) light microscopy but all utilize the principle of optical section recording, i.e. the 3-D image is a sequence of two-dimensional (2-D) images called optical sections. The optical sections are particular focal planes formed within the thick specimen and usually correspond to the conventional image projections recorded in a light microscope, referred to as x,y projections. The optical sections are recorded for a sequence of focus- or z-positions. This “stack” of 2-D images is the data set for the 3-D image. If quantitative analysis is to be performed on the 3-D images, the choice of the z-dimension increment between 2-D images is especially important, and its value may be more or less critical depending on the analysis algorithm used. A reasonable starting value for this dimension is the depth-of-field of the objective lens, but the actual value may have to be smaller to optimize the image analysis or larger to decrease the influence of photobleaching. The most photostable dyes should be selected and the specimen should be mounted in index-of-refraction matching media with an antioxidant.

The image resolution in all three-dimensions is determined by the 3-D point-spread-function (psf), and as a rough rule of thumb the z-resolution is degraded by a factor of 3 relative to the x,y resolution. To achieve or at least approach isotropic resolution the 3-D image can be deconvolved. Figure 1 shows the 2-D maximum value projection of a 3-D image of a cultured glial cell dual labeled for actin and vinculin before and after deconvolution. The actin fibers and vinculin focal contacts are more clearly resolved after deconvolution. Although a single cultured cell might traditionally be considered a thin object, it is really a thick object if the desired spatial resolution is less than the thickness of the cell. It is desirable to image as deep into a thick object as possible to maximize the tissue volume sampled. However, it has been shown that the image signal decreases with depth into the specimen. Figure 2 demonstrates this effect in a 3-D image of the nuclei of the rat hippocampus that have been labeled with the fluorescent Schiffs reagent acriflavine. In the x,y projection, it is not clear why some nuclei are dimmer than others, but the x,z projection shows that the dimmer ones tend to be deeper in the section. It has been shown that this depth dependent signal attenuation follows the form of an exponential function.

This presentation will describe a common core set of widely applicable image analysis techniques for automated quantitative analysis of volumetric microscope image data. Volumetric (as distinct from stereoscopic) three-dimensional (3-D) Microscopy is a rapidly maturing field offering the ability to image thick (compared to the depth of field) specimens using a variety of instrumentation techniques, and producing arrays of brightness values in three spatial dimensions. Also well developed are methods to correct the acquired images for a variety of physical effects including blur and attenuation.

Commonly, what is of interest is the best-possible visualization of thick specimens. The next step, increasingly being considered in view of growing computational resources, and progress in image analysis techniques, seeks to quantify many of the processes and effects being studied. In some mainstream fields, such quantitation is essential. For instance, various assays for substance testing in pharmaceutical and chemical industries involve quantitative end points. As an illustration, the Draize assay for ocular irritancy testing of drugs and biochemical products for human use requires counting of live and dead cells that stain differently. Another example is the mouse lymphoma test that requires a 3-D counting of bacterial colonies. Neurobiological assays require morphometry, as well as quantification of changes in neurons as a function of time and various applied stimuli such as drugs, heat, and radiation. Angiogenesis assays require quantification of changes in vascular morphometry. Computerized image analysis is a powerful tool for extracting quantitative data from 3-D images for statistical analysis.

One class of biological structures that has always presented special difficulties to scientists interested in quantitative analysis is comprised of extended structures that possess fine structural features. Examples of these structures include neuronal spiny dendrites and organelles such as the Golgi apparatus and endoplasmic reticulum. Such structures may extend 10's or even 100's of microns, a size range best visualized with the light microscope, yet possess fine structural detail on the order of nanometers that require the electron microscope to resolve. Quantitative information, such as surface area, volume and the micro-distribution of cellular constituents, is often required for the development of accurate structural models of cells and organelle systems and for assessing and characterizing changes due to experimental manipulation. Performing estimates of such quantities from light microscopic data can result in gross inaccuracies because the contribution to total morphometries of delicate features such as membrane undulations and excrescences can be quite significant. For example, in a recent study by Shoop et al, electron microscopic analysis of cultured chick ciliary ganglion neurons showed that spiny projections from the plasmalemma that were not well resolved in the light microscope effectively doubled the surface area of these neurons.

While the resolution provided by the electron microscope has yet to be matched or replaced by light microscopic methods, one drawback of electron microscopic analysis has always been the relatively small sample size and limited 3D information that can be obtained from samples prepared for conventional transmission electron microscopy. Reconstruction from serial electron micrographs has provided one way to circumvent this latter problem, but remains one of the most technically demanding skills in electron microscopy. Another approach to 3D electron microscopic imaging is high voltage electron microscopy (HVEM). The greater accelerating voltages of HVEM's allows for the use of much thicker specimens than conventional transmission electron microscopes.

Not everyone can afford to own and operate a scanning electron microscope. Everyone can, however, access images and instruments in a variety of ways, depending on the objective. Remote access capability is a result of recent developments in desktop computers, software, and the World Wide Web. Some of these developments and some suggestions for future resource development will be reviewed here.

With the arrival of commercially available, digitally controlled SEMs in the 1980s, it became possible to control microscope operation with a desktop computer and the appropriate software. By connecting a desktop computer to the microscope via the Internet or dedicated data lines such as ATMs, remote control of microscopes became a reality. Although analog microscopes are not as readily controllable over the Internet, their video data stream is easily captured, digitized, and dispatched to remote observers using low-cost consumer video conferencing programs. These approaches enable users at one location to observe their samples in a microscope located elsewhere. However, transfer rates over the Internet fluctuate wildly depending on network traffic, at times making remote access or control almost impossible. Dedicated data lines ensure a relatively stable transfer rate, but at a significantly higher cost.

The kaolinite-mullite reaction series by heating has been controversial since the proposal of a new transformation mechanism. Recently energy-filtered electron diffraction data obtained from the specimen furnace-heated at 920 °C indicated that the spinel-type phase coexisted topotactically with metakaolinite. On the other hand, mullite first appeared from the specimen furnace-heated at 940 °C without showing any crystallographic relationship to the parent metakaolinite.

Direct beam heating experiments of the 920 °C-heated specimens using an energy-filtering TEM supported the previous results and ideas. Fig. 1 displays topotactic coexistence of metakaolinite and the spinel-type phase (spots in hexagonal arrangement) before beam heating. Electron beam heating gave rise to a noticeable change in the diffraction pattern, that is, the formation of mullite rings and enhancement of the diffuse intensity around the direct beam shown in Fig. 2. Comparison of EDS data for the specimen before beam heating and after beam heating (Fig. 3) indicates considerable increase of Si and O peaks by beam heating.

Aragonite (CaCO3) is one of the principal carbonate minerals utilized by metazoans to form shells, tests and other skeletal elements. It may also be inorganically precipitated under certain geological conditions. Aragonite is unstable in meteoric diagenetic environments (relative to its polymorph calcite), and is prone to dissolution and/or replacement by other minerals (usually calcite). The mobilization of Ca2+ and CO32− during aragonite alteration is largely responsible for early diagenetic cementation and the formation of limestone from unconsolidated carbonate sediment. Our study has been investigating the very earliest stages of aragonite alteration. To date, we have limited our work to bivalves of the genera Donax, Chione and Austrovenus collected from, respectively, southern Alabama, southwestern Florida and eastern North Island, New Zealand. Chione and Austrovenus are closely related genera which should permit direct comparison of results. The Florida and New Zealand study locations were chosen because they were near fossil localities containing identical Tertiary-aged specimens that had been subjected to more prolonged alteration. Our results confirm that initial aragonite alteration in meteoric environments is driven by dissolution. Outer surfaces begin to dissolve very rapidly (laboratory experiments suggest that this may occur after only a few months of exposure to meteoric water); leading to a chalky consistency along the outer extremities of the shell. Simultaneous selective leeching of some trace elements (notably Sr2+) also occurs. More prolonged dissolution results in progressive loss of mass and Sr2+ as well as penetration of the chalky “zone” into the interior of shells. Highly altered shells consist almost entirely of chalk.

Atomic Force Microscopy (AFM) imaging of a komatiite specimen from the Morro do Ferro Greenstone Belt, M.G, Brazil, has revealed thin (0.2 - 2.0 micron in width), randomly oriented olivine crystals (fig. 1a,b), of prismatic shape. The length of such crystals is highly variable, ranging from less than 1 μm to more than 10 μm. The randomly oriented crystals typically occur in three or more distinct directions, indicating that the spinifex texture is reproduced at the micron-scale.

In addition, typical skeletal crystals were observed (figure 1c,d) with width of the order of one micron. Figure 1d is the sketch representation of the skeletal crystal shown on figure 1c Figures 1c,d also show that skeletal crystals sometimes branch out thinner crystals, indicating that nucleation also occurs at the surface of previously crystallized minerals, besides generating isolated new crystal seeds.

It is worth noting that the olivine crystals are characterized as low topographic regions on the AFM images. What distinguishes them from polishing scratches is the fact that scratches typically do not mimic crystal shapes, especially skeletal, or fabrics which are readily seen on different scales, i.e., petrographic sections or hand-specimens.

A number of cave systems host colorful deposits of what has been termed “corrosion residue” (CR), material that appears to be the breakdown product of bedrock minerals. The CR may be red, pink, orange, ocher, brown gray, or black and usually occurs in a variety of places within caves (ceilings, walls, tops and sides of boulders, etc.). Geologists have hypothesized that CR is the long-term result of upwelling corrosive air. However, discovery of evidence of microbial activity has led to a complimentary explanation that microbes could be active participants in the production of the corrosion residue. These deposits have been cursorily examined in several caves, including Jewel Cave (SD), Lechuguilla and Spider Caves (NM), and Cueva de Villa Luz (Tabasco, Mexico) using microscopy techniques of SEM and TEM, along with EDS and WDS analysis.

In all cases, the CR is a complex mixture of iron and/or manganese oxides, clays, quartz, and corroded bedrock material. In several cases, rare earth element (REE) phosphate minerals and other unusual minerals have been observed. The REE minerals are believed to result from recrystallization of apatite present in the bedrock; rare earth elements are commonly present in trace amounts within the apatite minerals. X-ray diffraction and bulk chemical analysis has shown that Fe3+ makes up the bulk if not all of the Fe-oxides in the CR while microprobe analysis of thin sections of the bedrock has found Fe2+ in dolomite as high as 700 ppm.

Within much of examined material, evidence of microbial life is present in the form of structures shaped like coccoid or filamentous bacteria. There is often a close association of presumptive bacteria with small dissolution pits in corroded fragments of bedrock minerals in the CR (Figure 1). In addition to the putative bacteria, the mineral residue often hosts small star-shaped minerals containing Fe-oxide (Figure 2).

In the early days of geochronology, attempts were made to date U-Th bearing rocks and minerals using the Pb-U-Th chemical method, but fundamental assumptions that all the lead was of radiogenic origin, and the system was closed, often were invalid. With the advent of the U-Th-Pb isotopic and common-lead methods of dating rocks and minerals, the chemical method was deemed clinically dead. However, several workers, for example, Montel et al., Suzuki and Adachi, have recently demonstrated new life for the chemical method. Using, the same theoretical basis as the chemical method, geologically reasonable dates often can be obtained by electron microprobe analysis of U-Th rich monazite (Ca-REE-U-Th phosphate). Other U- or Thrich minerals sometimes can be used to obtain microprobe U-Th-Pb dates, e.g., zircon, xenotime, uraninite.

Microprobe U-Th-Pb dates of uraninites from the McAllister Ta-Sn pegmatite, Coosa County, Alabama, are reported here. The McAllister pegmatite is a NW-SE dike-like body in the western part of the Rockford granite pluton. Polished grain mounts were made from screened table runs of heavy mineral separates. Wodginite (Mn, Sn, Nb, Ta-oxide), tantalite-columbite, zircon, cassiterite comprise most of the grains in the samples. Uraninite occurs as inclusions, generally <0.01 mm, in these grains (Figure 1).

U, Th, Pb concentrations were measured using a JEOL 8600 Superprobe. Operating conditions were 100 nA, 20 kV, and count times up to 200 seconds for peak plus background. The U Mβ, Th Mα, and Pb Mα peaks were measured. Standards were UO2, ThO2, and PbS.

An understanding of the solid-phase thermodynamics and aqueous speciation of aluminum is critical to our ability to understand and predict processes in a wide variety of geologic and industrial settings. Boehmite (AlO(OH)) is an important phase in the system Al2O3-H2O that has been the subject of a number of structural and thermodynamic studies since its initial synthesis [1] and discovery in nature [2]. Unfortunately, it has long been recognized that thermogravimetric analysis (TGA) of both synthetic and natural boehmite samples (that appear well crystallized by powder XRD methods) yields significant excess water - typically losing 16-16.5 wt. % on heating as compared with a nominal expected weight loss of 15.0 wt. % [3,4]. The boehmite used in our experiments was synthesized hydrothermally from acid-washed gibbsite (Al(OH)3) at 200°C. Powder XRD and SEM examination showed no evidence of the presence a contaminant phase. The TGA patterns do not suggest that this is due to adsorbed water, so a structural source is likely. We therefore undertook to examine this material by TEM to clarify this phenomenon.

Boehmite is orthorhombic (a = 0.0285nm, b = 0.1224nm and c = 0.0365nm, Amam). The crystals were tabular with major surfaces normal to <010>. Simple powder dispersals onto holey carbon films typically resulted in b-axis orientations parallel to the electron beam. To view other orientations, specimens were mulled in M-Bond epoxy, pressed between plates of single-crystal Si (aligning the boehmite tablets parallel to the Si plates) while the epoxy cured. Electron transparent thin foils normal to the silicon plates were produced by argon ion milling techniques. Sample stability in the electron beam was dramatically improved by cooling to −140°C using an 2 cold stage.

This paper will cover the history of the first steps of electron probe microanalysis. I was not personally involved with the electron microprobe at the very beginning, although I have been fully involved in the field from 1955. Nevertheless I have interesting information about the heroic times - when, just after the World War, Raimond Castaing was preparing for his doctoral degree.

In January 1947, R. Castaing, graduate in physics, joined the Institute for Aeronautical Research (ONERA), recently created by the French government. In the Materials Science Department, he was very lucky to be endowed with two electron microscopes, very exceptional equipment - a real luxury in 1948. The first one, an American RCA instrument was devoted to metallurgical studies. The second one, build by a French company, was cannibalized by Castaing to produce a fine electron beam to perform point chemical analysis. The idea was suggested to him by Andre Guinier, a famous x-ray crystallographer (Guinier-Preston zones, discovered in 1939) as the best way to solve identification problems in metallography. Castaing was not so enthusiastic about this proposal; the story seemed to him too simple, so simple, he thought, that it was quite surprising that nobody had done it before; but he soon had to face many physical problems that made him understand why many people probably failed, especially lens aberrations that had to be corrected to produce the intense, fine beam of electrons. Another question soon arose about x-ray detection. It was easy to detect the continuous background, but not very informative. Finally Guinier lent Castaing one of his precious quartz crystals (specially cut and ground, of the Johanson type); it was adjusted on a small spectrograph that fitted the outside of the main column (safety rules were in their infancy!). In the first days of 1949, he was able to measure characteristic x-ray emission from a l-μm, 4 nA electron probe. Considering the poor conditions in France after the terrible war, the rapidity with which this result was obtained is quite remarkable.

The early days of the electron microprobe were characterized by the variety of designs emerging from different laboratories in Europe, the United States and the USSR. Notable amongst these was that of Castaing in 1954, which employed a magnetic lens in combination with an optical microscope for viewing the sample and positioning the electron probe on the desired point for analysis. The X-ray emission was analysed by two high resolution spectrometers having their axes in the same plane as the electron-optical axis, and with their foci accurately set to coincide with the point of impact of the electron probe. This was a design well suited to point analysis by high resolution X-ray spectroscopy and formed the basis of the first Cameca instrument (Fig 1a).

By contrast, work by Duncumb in the Cavendish Laboratory in Cambridge started with the object of scanning the electron probe over the sample, in order to image the surface in terms of its characteristic X-ray emission. This required a strong lens to give a high current into a finely focused electron probe (Fig. 1b). The first element maps were demonstrated in 1956, and led to the design of Cambridge Instruments’ Microscan, intended as a metallurgical instrument, in conjunction with D.A. Melford of Tube Investments Research Laboratories.

Meanwhile, Long, also in Cambridge, was pioneering applications to mineralogy, and built an instrument for studying conventional slide-mounted rock samples, which could be viewed optically while analysis was in progress (Fig. 1c). This made use of a weaker probe-forming lens, with space for an inclined sample to be viewed in transmitted light. The slim design of the lens allowed it to be partially enclosed in the spectrometer, which received X-rays leaving the sample at a high angle to the surface - a benefit carried through into the Cambridge Geoscan.

The main initial source of information in the USA concerning the electron probe was a meeting at the Naval Research Laboratories in February 1958, followed in 1960 by a summer school at MIT organized by Prof. Norton and R. Ogilvie. Further visits to the USA and publications in English by the inventor of the instrument, R. Castaing, also contributed to the attention given here to the new technique. Since numerous research laboratories in the USA were keenly interested in the new device, the development of instruments and the contributions to its use were considerable. The relatively small number of active researchers in the field rendered personal connections and cooperation possible and attractive.

Before the availability of American-made commercial microprobes, many investigators built their own instruments. Most of them were not trained as instrument builders, and the usefulness of the resulting devices was somewhat limited. These improvisations in instrument construction ceased once commercial microprobe manufacturers entered the field. Yet, in the process, much was learned and the American investigators contributed significantly to the theory of microanalysis. We realized that, with the limited calculation facilities then available, the complex interactions leading to the x-ray signal had to be treated in simplified ways, and we described ‘approximations’ rather than ‘laws’ in our efforts to understand and quantitatively describe the physical facts. We also came to realize that even where the physical processes were well known, we often lacked the knowledge of the physical parameters required for quantitation and the effect of their uncertainties on the accuracy of the analytical results. Another area in which advances were needed, and soon achieved, was the application of the technique to a wide range of technical and scientific problems. The interaction between manufacturers and users greatly stimulated improvements in the instrument design.