We will review the results of structural approaches used in our laboratory for evaluating the roles of calcium (Ca2+) and the small GTPase, RhoA, in normal and pathological cell signaling.

An early method, using strontium (Sr) as an electron opaque marker of mitochondrial Ca, was first used to localize this “Ca2+ surrogate” in the sarcoplasmic reticulum (SR) of smooth muscle. Subsequent development of electron probe X-ray microanalysis (EPMA) and electron energy-loss spectroscopy (EELS) allowed the direct and quantitative detection of intracellular Ca in smooth, skeletal and cardiac muscles. The major conclusion of these studies was that, in all muscles, the SR is the major intracellular sink/source of activator Ca2+. The high ratio of total/free cytoplasmic Ca2+ indicated the presence of significant cytoplasmic Ca buffers not only in striated, but also in smooth muscles, and EPMA of frog skeletal muscle showed that the time course of changes in cytoplasmic Ca during relaxation was consistent with the off-rate of Ca2+ from the Ca2+-buffer, parvalbumin.

Imaging cells using a variety of microscopy techniques has generated information about the organization of cells and subcellular structures that is the foundation for understanding cell function. Invaluable information about the dynamics of cells has been obtained from a variety of light microscopy techniques, and the exquisite structural details of cellular organization have been obtained from electron microscopy. Each of these approaches, however, has its limitations. The resolution provided by light microscopy is limited by the wavelength of light, whereas the specimen preparation required for examining cells using electron microscopy is extremely tedious and time consuming. We show that soft x-ray microscopy can bridge the gap between these two forms of microscopy by providing a method for examining whole, hydrated cells up to 10 um thick at 25 nm resolution. Examination of rapidly frozen cells provides information that closely approximates that seen in living cells.

The activity of myosin II from vertebrate smooth and non-muscle cells is controlled by phosphorylation of the regulatory light chain (RLC). Smooth muscle heavy meromyosin (HMM) is a truncated double-headed myosin molecule that is soluble at physiological ionic strength. Subfragments of myosin containing two heads retain phosphorylation dependent regulation but single headed subfragments do not and are always “In”(Cremo et al., 1995; Trybus et al., 1997) thereby implicating head-head interactions as a fundamental feature of regulation. We have used a positively charged lipid monolayer to obtain 2-D crystalline arrays of both the unphosphorylated, inactive form (I-form) and thiophosphorylated, activated form (P-form) from chicken gizzard smooth muscle HMM obtained from a Baculovirus expression system.

A comparison of averaged 2-D projections of both forms in negative stain at 2.3 nm resolution reveals distinct structural differences (Wendt et al., 1999). The two crystals have p2 symmetry but vastly different unit cell dimensions.

Muscle contraction and the actomyosin ATPase that drives the contractile process are switched on and off by changes in sarcoplasmic free Ca2+ -concentration. In skeletal and cardiac muscles, on-off switching is mediated by the actinassociated protein tropomyosin and by the troponin complex. While the details of this mechanism are still subject to debate, it is well-accepted that tropomyosin strands move to sterically block and unblock myosin binding sites on actin, thereby controlling actomyosin ATPase and consequently contraction. It is also well known that the Ca2+- dependency of the movement of tropomyosin on actin is governed by troponin.

As a means of studying tropomyosin movement and the influence of troponin, we have used cryo-EM, negative staining and 3-dimensional helical reconstruction to define the positions of tropomyosin and troponin on thin filaments. We examined various preparations of native isolated filaments and filaments reconstituted with wild-type and mutant proteins.

In the 19th century, the microscope was the principal tool of biologists. The ordinary light microscope is designed to work on specimens that absorb light, but most unstained biological specimens do not absorb light appreciably and almost nothing is visible when such a specimen is viewed with the full aperture of the objective illuminated. However, they do show appreciable differences of refractive index, corresponding to variation in the concentration of solids, and the 19th century microscopists understood how to interpret the image when these were made visible by reducing the illuminating aperture and going slightly out of focus. If the objective is brought closer to the specimen (low focus), regions with high refractive index appear dark (positive image), and it became customary to use this setting, as in Fig. l (upper). As a result, the A bands of striated muscle fibres, with refractive index higher than the I bands, came to be known as the dark bands, although they appear bright with high focus.

Adult frog phasic skeletal muscle cells have slow inward calcium current (ICa) (Stanfield, 1977) carried through L-type voltage dependent calcium channels. It has been suggested that ICa may play a role in E-C coupling (Cota & Stefani, 1981, 1989). However, phasic skeletal muscle cells contract for several minutes after the extracellular Ca2+ concentration ([Ca2+]o) is lowered to <10-8 M (Armstrong et al, 1972). Therefore, extracellular Ca2+ (Ca2+o) is not essential for contraction in these fibers. It has been also shown, by blocking Ica that Ica is not essential for triggering contraction (Gonzalez-Serratos et al., 1982). These results have led to the conclusion that Ica has no obvious role in E-C coupling in adult amphibian phasic skeletal muscle. The question arises then as to what is the biological role Ica in phasic skeletal muscle? We have observed that embryonic skeletal muscle cells are capable of contracting during the first day of development in culture (Cordoba-Rodriguez, et al., 1996), before the T-system and the sarcoplasmic reticulum (SR)may have fully developed (Flucher, et al., 1993).

Experiments were carried out to investigate excitation-contraction coupling in vertebrate skeletal muscle fibers. Single-dissected twitch fibers of frog muscle were injected with flourescent calcium (Ca) indicators, and fluorescence intensity (F) and changes in intensity (ΔF) were recorded during various stimulation protocols (16-22 °C). In fibers activated by an action potential, the low-affinity indicator furaptra was used to estimate the spatially-averaged change in myoplasmic free calcium concentration (ΔC[a]; peak value, 15-20 μM; time to peak, 4-5 ms). Δ[Ca], in combination with a kinetic model, was used to calculate the change in concentration of calcium bound to the principal myoplasmic Ca buffers (ATP, troponin, parvalbumin). The rate of Ca release from the sarcoplasmic reticulum (SR) was obtained from the time derivative of the estimated change in total myoplasmic calcium concentration, Δ[CaT] (= Δ[Ca] + Δ[CaATP] + Δ[CaTroponin] + Δ[CaParvalbumin]).

Muscle contraction is activated by delivery of calcium from the internal membrane system of muscle cells, the sarcoplasmic reticulum (SR), to the myofibrils. Muscle relaxation ultimately depends on the sequestering of calcium back into the SR. In general, skeletal muscle is the most efficient in holding on to its calcium and calcium movements across the surface membrane are minimal during muscle activity. Cardiac muscle is less efficient and its activity is accompanied by significant movements of calcium across the surface membrane. Understanding of the calcium movements during muscle activation has relied heavily on structural information using the electron microscope and Lee Peachey was at the forefront in establishing structure function correlations. He talked the physiologists language and thus caught their attention.

Four stages can be detected in the contribution of EM to the unraveling of the calcium story. First the membrane systems involved were identified, and quickly sorted into two types on the basis of structural characteristics dictating specific functions.

It is proposed to build two high-performance general-user instruments at Daresbury, Cheshire, where the present U.K. synchrotron facility is located. The instruments can be used on-site, where accommodation for visitors is already available. They can also be used remotely, via the internet. The features of the instruments are: aberration-corrected optics, voltage-stabilised electron spectrometer, and multi-specimen stages to facilitate rapid throughput of routine work. We anticipate that a sub-Angstrom probe size will be available for incoherent HAADF imaging, and PEELS with an energy resolution better than 0.3V for losses up to 2kV. The operating voltage will be l00kV.

The new instruments will allow better analysis of continuing problems. Anticipated developments are as follows:

1. Study of helium bubbles in reactor materials: Recently, accurate quantification of helium has been achieved and a quantitative explanation has been given for the ‘blue shift’, that is, the increasing threshold energy for the Is to 2p transition due to quantum confinement of the excited state with increasing gas pressure.

Electron probe formation in a scanning transmission electron microscope (STEM) has two properties that maximize the benefits of spherical aberration correction: the smallest and brightest probes are formed when all the geometric aberrations are set to zero, and the size of the probe is not greatly affected by the presence of chromatic aberration. This contrasts with the case of conventional, fixed-beam TEM (CTEM), in which optimized phase-contrast imaging demands a non-zero spherical aberration coefficient (Cs), and chromatic aberration constitutes a major resolution limit. As a result, a consensus is presently emerging that the benefits of aberration correction will be felt most strongly in STEM.

Our efforts in Cs-corrected STEM have progressed from a proof-of-principle Cs corrector [1] to an optimized second-generation design [2]. The corrector in both cases is of the quadrupole-octupole type. The second-generation corrector uses separate quadrupoles and octupoles, and concentrates on maximizing the octupole strength.

It is the presence of local variations in atomic and electronic structure that often gives rise to intriguing properties of materials. Control of material properties then rests on detection of the local variations in the materials, determination of the origin of those variations and then understanding how such variations can be controlled to achieve a desired functionality. Recent examples using STEM with an atomic sized probe (∼2.2 Å) include studies of the bonding at grain boundaries in nickel rich Ni3Al doped with boron. EELS measurements revealed changes in the d-hole density-of-states that were used to estimate changes in the electronic contribution to the grain boundary energy. These changes in grain boundary strength correlated with a change in fracture from an intra- to an inter-granular mode. In another example, the observation of metal induced gap states at Cu/MgO interfaces provided evidence that the dipole moment across that interface was relatively low.

In making quantitative measurements using electron micrographs we must regard the electron microscope as being an information channel. The information about the object is both in some way transformed by the image forming process, and information is lost through the existence of a spatial resolution limit and the addition of noise. To make quantitative measurements, we need to take account of all these effects.

Great progress has been made in making quantitative measurements from high-resolution transmission electron microscope (HRTEM) images (for example see Ref. [1]). However, the simulation of HRTEM images requires a full dynamical electron scattering calculation in addition to computing the effects of the objective lens aberrations. Although such calculations can provide excellent agreement with experimental data, the calculation times involved restrict the number of trial structure models that can be used, with the danger that such trial and error methods might miss the true object solution.

The demonstration of an aberration corrector for the STEM promises enormous improvements in the contrast and signal to noise ratio of Z-contrast images, with similar benefits for atomic column EELS. Here we show that the limiting resolution for a zone axis crystal will become not the probe, as in the case of isolated atoms, but the Is Bloch states. In fact, the Z-contrast image becomes a direct image of the Is Bloch states with limiting intensities for large thicknesses roughly proportional to Z The potential benefits for the (STEM) appear to far exceed those for the conventional TEM. Some of these benefits are intrinsic to incoherent imaging: the lack of interference artifacts and the potential factor of two improvement in image resolution were first pointed out by Lord Rayleigh. This improved resolution has been demonstrated by the achievement of sub-ingstrom information transfer in the VG Microscopes HB603U, and the resolution advantage will still apply after aberration correction.

Semiconductor devices are rapidly heading towards nanometer sizes, with dielectric gate oxides already in the 2-3nm thickness range and transistor channel lengths of order 10-20nm. There is good reason to believe, therefore, that physical limits imposed by atomic level granularity will dominate operation of semiconductor devices in the future. Thus, recent work has identified a physical limit for the thickness of SiO2 in order to maintain its insulating character. [1] On the other hand, new opportunities are created, based on new behavior at the atomic level. In the presence of very high local electric fields, for instance, the local electronic structure can change from insulating to conductive, forming a very small, very fast “Mott” transistor. [2] In a single molecule having a localized electronic level which is positioned well with respect to a conducting environment, single electron transistor operation may be possible at room temperature. [3]

Sub-0.2 nm probes can now be readily obtained on Schottky field-emission microscopes[1]. However environmental instabilities are proving to be the limiting factors for atomic resolution spectroscopy and distortion-free annular-dark field imaging, as a result of the long acquisition times (comparable to those required for inline holography[2]), and from the serial nature of the scanning system where instabilities result in image distortions rather than reductions in contrast. Troubleshooting the two most common environmental problems are discussed here.

Electromagnetic interference can cause beam deflections in both the scanning system and the spectrometer [3](< 0.3 mG r.ms for 0.3nm, < 0.2 mG for 0.2 nm). These are most easily dealt with before the machine is installed, as substantial rewiring may be necessary. There is little that can be done about quasi-DC fields, such as from elevators and nearby trains and buses. Major sources of AC electromagnetic interference are unbalanced electrical loads.

Commercial TEM/STEMs are ill designed for quantitative X-ray microanalysis of thin foils. There have been no fundamental advances in their design since the first instruments appeared in the mid- 1970s. These instruments had thermionic sources with useful probe sizes of ∼10 nm, small (∼ 0.1 sr) detector collection angles, illumination systems giving serious levels of stray radiation, substantial hydrocarbon and water-vapor contamination due to the poor vacuum (∼10-5Pa) and sliding o-ring seals on unstable side-entry goniometer stages. The instruments lacked a direct measure of the probe current at the specimen. Often a 60 s accumulation time resulted in enough drift or contamination that the microanalysis was not trustworthy and spatial resolution was compromised. Today's modern TEM/STEMs, apart from replacement of thermionic source with a FEG, are little better.

Dedicated STEMs however, have invariably offered better X-ray performance. The first VG HB 5 DSTEM became available, also in the 1970s, and the X-ray performance improved through to the latest design (the VG HB 603) in the mid-1990s.

The origin of electrically active grain boundaries in perovskite oxides and related materials remains controversial. The stoichiometry of the grain boundary core structure and the role of oxygen vacancies are the key issues involved. Previous results have given no indications of any non-stoichiometry at SrTiO3 grain boundaries, despite the fact that SrTiO3 is the system where atomic column EELS resolution has been demonstrated [1], and where atomic resolution images of the core structures have been obtained by Z-contrast imaging [2,3]. Here we show EELS spectra from individual dislocation cores in an 8° [100] tilt grain boundary in SrTiO3 that show significant oxygen depletion.

The low angle grain boundary with well-separated dislocation cores makes it possible to study individual cores with high energy resolution in EELS. Previous work either averaged over all structural units in the grain boundary, sacrificing spatial resolution [2,3], or, used a very low probe current to obtain the highest spatial resolution [1].

A Hitachi HD-2000 dedicated STEM with a cold field emission source was installed last fall at the Lucent Technologies Semiconductor Fab site in Orlando. The close relationship between the Orlando arm of Lucent Technologies and the University of Central Florida has and will allow this instrument to be evaluated for its usefulness in two roles: Support of the development and fabrication needs of a semiconductor fabrication facility and the research needs of University based Materials research.

This Hitachi instrument was specifically designed to meet the needs of the semiconductor industry. Sample holder compatibility with the Hitachi Focused Ion Beam (FIB) allows rapid swapping between the two tools. A large solid angle Energy Disperse X-ray Spectrometer (EDS) supports increased resolution and sensitivity of X-ray analysis. Direct digital acquisition speeds image and data transfer to the end user. All these and other features speed the turn around time and usefulness for manufacturing line feedback.

The Perovskite structured ceramic (Lax,Sr1-x)(Fey,Cr1-y)O3-δ being developed for applications in oxygen transporting membranes. The permeability of this material is limited by the number of free ions, point-defects (oxygen vacancies) and electrons in the bulk. As any ordering of these unbound particles will restrict their mobility one key issue for controlling the membrane efficiency is the formation of ordered oxygen vacancies. In particular it is very likely, that at elevated temperatures ordered micro-domains progressively grow and asymptotically reach a stable equilibrium concentration. This is consistent with the observations of Kruidhof that below a specific order-disorder transition temperature equilibrium times of 30-40 h are required to attain steady-state conditions, irrespective of the thermal history of the sample.

We investigate the formation of ordered vacancies in (Lax,Sr1-x)(Fey,Cr1-y)O3-δ by atomic resolution Z-contrast imaging and electron energy-loss spectroscopy (EELS) using a 200 keV STEM/TEM JEOL2010L with a post column Gatan Image Filter (GIF).

Grain boundaries in yttria-stabilized cubic zirconia (YSZ) are believed to have positively charged grain boundaries with compensating negative space charge layers at ambient temperatures and pressures [1]. The space charge region is formed by equilibrium segregation of point defects to the vicinity of the grain boundary to establish local charge neutrality. The point defect distributions, calculated from analytical space charge models [1] show that the negative space charge region is formed by a depletion oxygen vacancies and an excess of substitutional Y atoms, indicated as'VO” and YZr’ in standard Kröger-Vink notation (see fig. 1). The grain boundary segregation, in turn, has important ramifications for the mechanical and electrical behavior of the material [1,2]. The present work addresses space-charge segregation in YSZ, specifically addressing variations in segregation with grain boundary misorientation. We have examined a range of symmetric tilt [001] YSZ grain boundaries since they allow us to systematically vary the misorientation while providing the opportunity for atomic scale analysis.