We have been researching the capability of atomic force microscopy to reveal nontopographic properties of tissue embedded in plastic and sectioned with standard electron microscopic techniques. We present topography and elasticity maps of plastic-embedded, thin sections of muscle tissue. The images show topography correlated with the normal repeating structure of the sarcomere. Elasticity mapping using force modulation revealed contrast between the actin- and myosin-rich areas. We attribute the observed contrast in elasticity to the difference in local concentrations of biological material in embedding plastic.

Micrometer sized particles have been studied to show that a high-quality transmission electron microscope (TEM) specimen can be produced, without the use of embedding media, from a site-specific region of chosen particles using the focused ion beam (FIB) lift-out (LO) technique. The uniqueness of this technique is that site-specific TEM LO specimens may be obtained from particles and from regions which are smaller than the conventional ∼10–20 μm × 5 μm × ∼0.1 μm dimensions of the LO specimen. The innovative FIB LO procedures are described in detail and TEM images of electron transparent specimens obtained from specific micrometer-sized particles are presented.

The development of the electron probe microanalyzer (EPMA) in Japan in the early 1960s, when Prof. R. Castaing visited Japan, is briefly outlined. In 1955, a review article was published by Prof. G. Shinoda in Oyobutsuri, the most popular journal in Japan, in which the EPMA was introduced. In 1957, a research group at the University of Tokyo started to develop an EPMA with a Grant-in-Aid for Developmental Research. Their research results led to the funding of a 2-year Grant-in-Aid for Cooperative Research Project (April 1960 to March 1962), which was chaired by Prof. Y. Sakaki. Prof. G. Shinoda who became the chairman of that project in April of 1962 led that group for another year until March of 1963. It was just after the start of the project that Prof. R. Castaing visited Japan in September of 1960 as a representative of the French Mission Culturelle. This visit gave a great push forward for the commercial development of EPMA instruments in Japan. The first three commercial EPMA instruments from Hitachi, JEOL, and Akashi Ltds. were installed in Tohoku, Osaka, and Waseda Universities in 1962, 1963, and 1964, respectively. Photographs of those first commercial EPMA systems, together with a brief description of the activities of the cooperative research projects, are presented.

Our recent observation that microtubules (MTs) are required for completion of division (abscission) led us to analyze MT organization during cytokinesis. Although many studies of MTs in sea urchin eggs have been done, computer-aided analysis of optical sections described herein reveals a new MT assemblage, which we call furrow MTs. This assemblage comprises bundles of MTs that lie in the cleavage furrow. Furrow MTs become apparent when the furrow has progressed approximately one-third of the way through the egg and persist to abscission. Furrow MTs are 8–24-µm long and arc across the base of the cleavage furrow. Acetylated tubulin is localized primarily in the furrow suggesting a distinct MT population. Three-dimensional analysis of optical sections suggests that furrow MTs are spatially distinct from midbody and astral MTs.

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.

Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.

The scattering cross section is the fundamental measure of the strength of a scattering interaction. All scattering in electron microscopy arises from the Coulomb interaction, and scattering cross sections, whether elastic or inelastic, will therefore all have common features. Simple forms of both elastic and inelastic cross sections are reviewed in the context of high resolution and analytical microscopy. Some recent developments, such as the calculation of Fano resonances in electron energy loss spectra of transition metals and rare earth elements are also discussed.

The environmental transmission electron microscopy (E-TEM) is a budding technique for in situ study of gas–solid chemical reactions with numerous applications. Recent improvements in the design have made it possible not only to obtain atomic level information but also the chemical information during the reaction by incorporating an imaging filter or electron energy-loss spectrometer to an E-TEM. We have been involved in modifying a couple of microscopes to incorporate environmental cells in order to convert them into E-TEMs. These microscopes have been used to obtain atomic level information of the structural and chemical changes during dynamic processes by in situ electron diffraction, high-resolution imaging, and electron energyloss spectroscopy. The applications include, but are not limited to, oxidation, reduction, polymerization, nitridation, dehydroxylation, hydroxylation, chemical vapor deposition, etc. We report recent developments in the design and application along with the limitations of an E-TEM.

A Mo-rich precipitate in an Fe-rich matrix and the heterophase interface bounding the precipitate have been examined on an atomic scale by three-dimensional atom-probe (3DAP) microscopy and transmission electron microscopy (TEM). The Mo-rich precipitate was generated by isothermal solid-state decomposition at 500°C of an Fe-15 at.% Mo-5 at.% V alloy. After this heat treatment, TEM reveals a characteristic modulated strain contrast structure. The precipitate has a composition of Mo-13.0 at.% Fe-4.9 at.% V as measured by 3DAP microscopy. The alloy decomposes at a nearly constant 5 at.% V, constituting a metastable tie-line between Fe-rich and Mo-rich bcc solid solutions. The formation of the equilibrium intermetallic phases is kinetically impeded. A local vanadium enrichment of 21 at.% is detected at the interface of the precipitate in a matrix region about 0.27 nm or 1–2 atomic layers in thickness. The corresponding value of the Gibbsian interfacial excess of vanadium is 3.3 ± 1.3 nm−2.

A method is described for the estimation of mean cell volume of phagocytosing cells. The cells are coembedded with yeast particles of known size. By combination of data obtained from morphometrical analysis of sections in both the light and transmission electron microscopes, an estimate of the ratio between cell volume and yeast particle volume is obtained. The method makes use of the dissector but does not require measurements on serial sections or knowledge of section thickness. Evaluation of the method was done by studying the effect of phagocytosis of latex beads on macrophage cell volume and surface area. It was found that the surface area of phagocytosing macrophages remained constant although the cell volume increased by 27%. Furthermore, in phagocytosing macrophages, the amount of membrane enclosing intracellular vacuoles decreased by 28%, but this loss of membrane was balanced by the appearance of membrane enclosing the phagocytosed latex beads. The method described here may prove useful for morphometrical studies on phagocytosing leukocytes, as well as on intracellular parasites located within these cells.

Sequence specific transcription factors are the predominant regulators of animal gene expression controlling nearly all biological processes. We are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within an entire organism. Pregastrula Drosophila embryos have been chosen because these embryos allow high resolution 3D optical imaging since they comprise a single layer of dividing cells surrounding a yolk sac. in addition, the transcription network controlling gene expression is well characterized in early Drosophila embryos[1], and is being further dissected by a multi-laboratory collaboration, the Berkeley Collaboration in Drosophila Genomics, which encompasses this work.

Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy (Figure 1). Intensity-based segmentation of the total DNA image[2] produces a nuclear mask which defines the nuclear boundaries, their location and the number of cells within the embryo (Figure 2). Presently, dilation of the nuclear volumes into their nearest-neighbours[3] is used to estimate the boundary of the cell (Figure 3) and superposition of these images produces a morphological mask defining each cell and its nucleus.

In situ transmission electron microscopy is an established experimental technique that permits direct observation of the dynamics and mechanisms of dislocation motion and deformation behavior. In this article, we detail the development of a novel specimen goniometer that allows real-time observations of the mechanical response of materials to indentation loads. The technology of the scanning tunneling microscope is adopted to allow nanometer-scale positioning of a sharp, conductive diamond tip onto the edge of an electron-transparent sample. This allows application of loads to nanometer-scale material volumes coupled with simultaneous imaging of the material’s response. The emphasis in this report is qualitative and technique oriented, with particular attention given to sample geometry and other technical requirements. Examples of the deformation of aluminum and titanium carbide as well as the fracture of silicon will be presented.

An increase in extracellular adenosine triphosphate (ATP) is arrhythmogenic in rat cardiac myocytes and extracellular ATP levels are elevated during cardiac ischemia. To gain insight into the mechanism by which the arrhythmic contractions are generated, we investigated changes in subcellular elemental content by electron probe microanalysis (EPMA) in isolated adult rat cardiac myocytes stimulated by the ATP analog, 2-methylthio- ATP (2-M-S-ATP). We also measured the effects of 2-M-S-ATP stimulation on myocyte cell shortening. In electrically stimulated myocytes, 2-M-S-ATP stimulation generated arrhythmic contractions and also increased the amplitude of cell shortening. However, only the arrhythmic contractions were reversed by 2-MS- ATP washout. EPMA of freeze-dried cryosections of rapidly frozen 2-M-S-ATP-stimulated myocytes showed increased cytosolic Na and Cl, decreased K, but no significant change in mitochondrial Ca upon 2-M-S-ATP stimulation. The arrhythmias were abolished upon 2-M-S-ATP washout, and the observed changes in cytosolic elemental content also reversed upon agonist washout, thus suggesting that the increased Na and Cl, and decreased K, are specifically associated with the ATP-dependent spontaneous contractile activity. The observed increase in intracellular Na upon 2-M-S-ATP stimulation may explain our observation of prolonged relaxation time of 2-M-S-ATP-stimulated contractions. This may be due to inhibition of Ca2+ efflux via the Na+ Ca2+ exchanger.

Castaing’s successful implementation and application of the electron probe microanalyzer in 1950 stimulated a flurry of development activity around the world. The later versions of this instrument represented a truly international effort, with significant contributions by scientists from Europe, Asia, and North America. If the probe-forming system of the instrument was its heart, the X-ray wavelength spectrometer was its soul. This article reviews some of the history of spectrometer developments—lthrough the “golden years” of microprobe development, namely the dozen or so years following the publication of Castaing’s thesis, to the present. The basic physics of spectrometer and crystal design is reviewed. Early experimental devices, such as those developed by Castaing, Borovskii, Wittry, Duncumb, and Ogilvie are reported. Examples of commercial spectrometers such as those by ARL, MAC, Microspec, and Peak are described. Recent developments such as the combination of grazing-incidence optics with flat crystal spectrometers are noted, and the properties and uses of doubly curved crystals are discussed. Finally, the continued development of doubly curved crystal configurations, such as the “Wittry geometry” for scanning monochromators, and point-to-point focusing diffractors for producing small monochromatic X-ray probes to provide improved detection limits for microanalysis are considered.

This is the first report on observation of phase change recorded marks by use of the scanning probe microscope (SPM). The amorphous mark and crystalline blank were clearly observed by surface potential mode (SPoM) that visualized the surface potential difference among phases or materials. SPoM observation has an advantage over conventional transmission electron microscope (TEM) observation in that the sample preparation is much easier, and that amorphous mark and crystalline blank are clearly distinguished. The possibility of a novel readout method of phase change disks, and the possibility of the novel medium with a surface potential detection method are also proposed.

The spherulitic and lamellar morphologies of melt crystallized isotactic polypropylene (iPP) have been investigated according to various crystallization temperatures and times. In this study, an appropriate chemical etching condition for the observation of iPP morphology by scanning electron microscopy (SEM) is determined. The optimum etchant formulation is 3 wt% potassium permanganate with the mixed solvent of sulfuric acid and phosphoric acid. The morphologies after etching reveal the microstructure of iPP clearly, such as the characterization of α/β spherulites and the different lamellar growth patterns.

This article will discuss the importance of Raimond Castaing’s thesis on the genesis of a nondestructive and truly quantitative microanalytical method that assisted the scientific community in moving forward in the development of microanalytical instruments. I will also share with you my recollection of the decades of improvement in the electron probe microanalyzer (EPMA), that has allowed us to reach our present level of instrument sophistication, and I will explore with you my thoughts on the future evolution of this technique. To conclude, I will present the current status of related microanalysis techniques developed under Castaing in Orsay in the 1960s, as Castaing’s interest in microanalysis was not limited to electron probe microanalysis alone.