A multicomponent Fe-Cu based steel is studied using atom-probe tomography. The precipitates are identified using two different methodologies and subsequent morphological and compositional results are compared. The precipitates are first identified using a maximum separation distance algorithm, the envelope method, and then by a concentration threshold method, an isoconcentration surface. We discuss in detail the proper selection of the parameters needed to delineate precipitates utilizing both methods. The results of the two methods exhibit a difference of 44 identified precipitates, which can be attributed to differences in the basis of both methods and the sensitivity of our results to user-prescribed parameters. The morphology of the precipitates, characterized by four different precipitate radii and precipitate size distribution functions (PSDs), are compared and evaluated. A variation of less than ∼8% is found between the different radii. Two types of concentration profiles are compared, giving qualitatively similar results. Both profiles show Cu-rich precipitates containing Fe with elevated concentrations of Ni, Al, and Mn near the heterophase interfaces. There are, however, quantitative disagreements due to differences in the basic foundations of the two analysis methods.

A real-space technique for finding structural information in atom probe tomographs, spatial distribution maps (SDM), is described. The mechanics of the technique are explained, and it is then applied to some test cases. Many applications of SDM in atom probe tomography are illustrated with examples including finding crystal lattices, correcting lattice strains in reconstructed images, quantifying trajectory aberrations, quantifying spatial resolution, quantifying chemical ordering, dark-field imaging, determining orientation relationships, extracting radial distribution functions, and measuring ion detection efficiency.

MSA's Megabooth (Booth # 1022) provides many services for meeting attendees. The Cyber Cafe offers free internet access. While you're waiting for a terminal, you can examine copies of recent microscopy books, for both adult microscopists and children. Sample DVDs of meeting tutorials are available for review. You can post a resume or job opening, or sign up for the exhibitor tutorials. Free copies of Microscopy Today are available. You can get information on MSA membership and MSA's technologist certification and undergraduate scholarship programs. Project MICRO, MSA's precollege outreach program and the Technologists' Forum both have information desks.

The nucleoid structure of an important human pathogen, Staphylococcus aureus, was dissected by atomic force microscopy (AFM). The nucleoids dispersed on a cover glass consisted of fibrous units with two different widths of 40 and 80 nm, a feature shared with those of Escherichia coli. On the other hand, cells exposed to an oxidative stress exhibited clogged nucleoids. A knock-out of mrgA (metallo regulated genes A) encoding a staphylococcal homolog of the nucleoid compaction factor (E. coli Dps) eliminated the compaction response to the oxidative stress and reduced the susceptibilities to H2O2 and UV irradiation. We also observed that the negative supercoiling of plasmids is increased by the oxidative stress. A possible interrelation between the helical density and the nucleoid compaction is discussed in relation to the oxidative stress response.

Atom probe tomography provides a chemical analysis of nanostructured materials with outstanding resolution. However, due to the process of field evaporation triggered by nanosecond high voltage pulses, the method is usually limited to conductive materials. As part of recent efforts to overcome this limitation, it is demonstrated that the analysis of thick NiO and WO3 oxide layers is possible by laser pulses of 500 ps duration. A careful analysis of the mass spectra demonstrates that the expected stoichiometries are well reproduced by the measurement. The reconstruction of lattice planes proves that surface diffusion is negligible also in the case of thermal pulses.

This article describes a fully automatic image analysis procedure for fast and reliable characterization of the activated sludge composition, that is, the floc and filament features. The algorithms developed for each of the analysis steps, that is, segmentation, object recognition, and characterization, are described in detail. Although the application range of the recognition method is a priori expanded by introducing a number of control parameters, the procedure proves to be intrinsically robust as it produces satisfactory results for a fixed set of parameter values for a wide variety of image types.

The application of scanning electron microscopy (SEM) and secondary ion mass spectrometry (SIMS) for characterization of mixed plutonium and uranium particles from nuclear weapons material is presented. The particles originated from the so-called Thule accident in Greenland in 1968. Morphological properties have been studied by SEM and two groups were identified: a “popcorn” structure and a spongy structure. The same technique, coupled with an energy-dispersive X-ray (EDX) spectrometer, showed a heterogeneous composition of Pu and U in the surface layers of the particles. The SIMS depth profiles revealed a varying isotopic composition indicating a heterogeneous mixture of Pu and U in the original nuclear weapons material itself. The depth distributions agree with synchrotron-radiation-based μ-XRF (X-ray fluorescence microprobe) measurements on the particle (Eriksson, M., Wegryzynek, D., Simon, R., & Chinea-Cano, E., in prep.) when a SIMS relative sensitivity factor for Pu to U of 6 is assumed. Different SIMS identified isotopic ratio groups are presented, and the influence of interferences in the Pu and U mass range are estimated. The study found that the materials are a mixture of highly enriched 235U (235U:238U ratio from 0.96 to 1.4) and so-called weapons grade Pu (240Pu:239Pu ratio from 0.028 to 0.059) and confirms earlier work reported in the literature.

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

Focused ion-beam milling has been applied to prepare needle-shaped atom probe tomography specimens from mechanically alloyed powders without the use of embedding media. The lift-out technique known from transmission electron microscopy specimen preparation was modified to cut micron-sized square cross-sectional blanks out of single powder particles. A sequence of rectangular cuts and annular milling showed the highest efficiency for sharpening the blanks to tips. First atom probe results on a Fe95Cu5 powder mechanically alloyed in a high-energy planetary ball mill for 20 h have been obtained. Concentration profiles taken from this powder sample showed that the Cu distribution is inhomogeneous on a nanoscale and that the mechanical alloying process has not been completed yet. In addition, small clusters of oxygen, stemming from the ball milling process, have been detected. Annular milling with 30 keV Ga ions and beam currents ≥50 pA was found to cause the formation of an amorphous surface layer, whereas no structural changes could be observed for beam currents ≤10 pA.

Nanoscale atomic clusters in atom probe tomographic data are not universally defined but instead are characterized by the clustering algorithm used and the parameter values controlling the algorithmic process. A new core-linkage clustering algorithm is developed, combining fundamental elements of the conventional maximum separation method with density-based analyses. A key improvement to the algorithm is the independence of algorithmic parameters inherently unified in previous techniques, enabling a more accurate analysis to be applied across a wider range of material systems. Further, an objective procedure for the selection of parameters based on approximating the data with a model of complete spatial randomness is developed and applied. The use of higher nearest neighbor distributions is highlighted to give insight into the nature of the clustering phenomena present in a system and to generalize the clustering algorithms used to analyze it. Maximum separation, density-based scanning, and the core linkage algorithm, developed within this study, were separately applied to the investigation of fine solute clustering of solute atoms in an Al-1.9Zn-1.7Mg (at.%) at two distinct states of early phase decomposition and the results of these analyses were evaluated.

The inaccessibility of osteocytes due to their embedment in the calcified bone matrix in vivo has precluded direct demonstration that osteocytes use gap junctions as a means of intercellular communication. In this article, we report successfully isolating primary cultures of osteocytes from chick calvaria, and, using anti-connexin 43 immunocytochemistry, demonstrate gap junction distribution to be comparable to that found in vivo. Next, we demonstrate the functionality of the gap junctions by (1) dye coupling studies that showed the spread of microinjected Lucifer Yellow from osteoblast to osteocyte and between adjacent osteocytes and (2) analysis of fluorescence replacement after photobleaching (FRAP), in which photobleaching of cells loaded with a membrane-permeable dye resulted in rapid recovery of fluorescence into the photobleached osteocyte, within 5 min postbleaching. This FRAP effect did not occur when cells were treated with a gap junction blocker (18α-glycyrrhetinic acid), but replacement of fluorescence into the photobleached cell resumed when it was removed. These studies demonstrate that gap junctions are responsible for intercellular communication between adjacent osteocytes and between osteoblasts and osteocytes. This role is consistent with the ability of osteocytes to respond to and transmit signals over long distances while embedded in a calcified matrix.

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

The M&M 2007 meeting will be preceded on Saturday and Sunday, August 5 and 6, with a Pre-meeting Congress entitled “Cryo-preparation for high-resolution cryo-SEM and cryo-TEM”. The two-day Congress will consist of “how-to” lectures, as well as demonstrations of a wide variety of cryo-EM instrumentation and techniques. The Pre-meeting Congress will share Sunday with full-day Short Courses on image analysis, immunogold labeling, biological confocal microscopy, failure investigation, surface analysis, and electron backscattered diffraction.

We introduce a simple preparation method for ultrathin carbon support films that is especially useful for high-resolution electron microscopy (HREM) of nanoparticles. Oxidized iron nanoparticles were used as a test sample in a demonstration of this method. The film qualities are discussed on the basis of electron-energy-loss spectroscopy (EELS) and image analysis techniques such as thickness maps and histograms. We carried out a comparison between the homemade and commercial film qualities. The relative thickness of the homemade support films was 0.6 times less than that of the commercial films, which was calculated from the EELS analysis, whereas the thicknesses of both carbon support films varied within about 3%. The percentage of the observable area was about 67 ± 7.6% of the support film. This was about twice as large as the commercial film (32 ± 9.3%). The HREM image of the sample prepared with our support film improved 9% in brightness and 15% in contrast compared with images obtained with the commercial support.

The derivation of a universal equation to compute the range of emitted X rays is presented for homogeneous bulk materials. This equation is based on two fundamental assumptions: the φ(ρz) curve of X-ray generation is constant and the ratio of the emitted to the generated X-ray range is equal to the ratio of the emitted to the generated X-ray intensity. An excellent agreement is observed with data obtained from Monte Carlo simulations of 200,000 electron trajectories in C, Al, Cu, Ag, Au, and an Fe–B alloy with boron weight fractions equal to 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.99, performed with the electron beam energy varied from 1 to 30 keV in 1-keV steps. When the ratio of the generated X-ray range to the photon mean free path is much smaller than one, the emission X-ray range is equal to the generated X-ray range, but when this ratio is much greater than one, the emission X-ray range is constant and is given by the product of the effective photon mean free path multiplied by the sine of the take-off angle.

Bacterially induced carbonate mineralization has been proposed as a new method for the restoration of limestones in historic buildings and monuments. We describe here the formation of calcite crystals by extracellular polymeric substances isolated from Bacillus firmus and Bacillus sphaericus. We isolated bacterial outer structures (glycocalix and parietal polymers), such as exopolysaccharides (EPS) and capsular polysaccharides (CPS) and checked for their influence on calcite precipitation. CPS and EPS extracted from both B. firmus and B. sphaericus were able to mediate CaCO3 precipitation in vitro. X-ray microanalysis showed that in all cases the formed crystals were calcite. Scanning electron microscopy showed that the shape of the crystals depended on the fractions utilized. These results suggest the possibility that biochemical composition of CPS or EPS influences the resulting morphology of CaCO3. There were no precipitates in the blank samples. CPS and EPS comprised of proteins and glycoproteins. Positive alcian blue staining also reveals acidic polysaccharides in CPS and EPS fractions. Proteins with molecular masses of 25–40 kDa and 70 kDa in the CPS fraction were highly expressed in the presence of calcium oxalate. This high level of synthesis could be related to the binding of calcium ions and carbonate deposition.

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Cryo-Preparation for High-Resolution Cryo-SEM and Cryo-TEM.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.